DNA encoding 5-HT4 serotonin receptors and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid molecule encoding a mammalian 5-HT 4  receptor and an isolated nucleic acid molecule encoding a human 5-HT 4  receptor, an isolated protein which is a mammalian 5-HT 4  receptor, an isolated protein which is a human 5-HT 4  receptor, vectors comprising an isolated nucleic acid molecule encoding a mammalian 5-HT 4  receptor, vectors comprising and isolated nucleic acid molecule encoding a human 5-HT 4  receptor, mammalian cells comprising such vectors, antibodies directed to the 5-HT 4  receptor, nucleic acid probes useful for detecting nucleic acid encoding a mammalian or human 5-HT 4  receptor, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a mammalian or human 5-HT 4  receptor, pharmaceutical compounds related to the human 5-HT 4  receptor, and nonhuman transgenic animals which express DNA encoding a normal or a mutant mammalian or human 5-HT 4  receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatments for alleviating abnormalities associated with a human 5-HT 4  receptor.

BACKGROUND OF THE INVENTION

[0001] Throughout this application various publications are referred toby partial citations within parenthesis. Full citations for thesepublications may be found at the end of the specification immediatelypreceding the claims. The disclosures of these publications, in theirentireties, are hereby incorporated by reference into this applicationin order to more fully describe the state of the art to which thisinvention pertains.

[0002] Molecular cloning efforts have provided primary amino acidsequence and signal transduction data for a large collection ofserotonin receptor subtypes. These include five cloned 5-HT₁-likereceptors, three cloned 5-HT₂ receptors, and one 5-HT₃ receptor. TheB-HT₁ subfamily includes: 5-HT_(1A) (Fargin, 1988; Kobilka, 1989),5-HT_(1B)/5-HT_(1Dβ) (Weinshank et al., 1991; Demchyshyn et al., 1992;Jin et al., 1992; Adham et al., 1992; Maroteaux et al., 1992; Voight etal., 1991), 5-HT_(1Dα) (Branchek et al. 1991; Hamblin and Metcalf, 1991;Weinshank et al., 1992), 5-HT_(1E) (Levy et al., 1992; McAllister etal., 1992; Zgombick et al., 1992) and 5-HT_(IF) (Adh.am et al., 1993).All five have been shown to couple to the inhibition of adenylatecyclase activity. The 5-HT₂ family includes the 5-HT₂ receptor(Pritchett et al., 1988), 5-HT_(1C) (Julius et al., 1989) and 5-HT_(2F)(Rat Stomach Fundus; Foquet et al., 1992; Kursar et al., 1992). Thesereceptors all couple to phosphoinositide hydrolysis. The 5-HT₃ receptoris a ligand-gated ion channel (Maricq et al., 1991).

[0003] Although this work represents enormous success, the absence ofmolecular biological information on the 5-HT₄ receptors, which have beenshown in native tissues to couple to the activation of adenylate cyclaseas a primary mode of signal transduction (Dumius et al., 1988; Bockaertet al., 1990), is apparent. In a previous copending application (U.S.Ser. No. 971,690, filed Nov. 3, 1992), we reported the cloning of thefirst mammalian 5-HT receptor that couples to the stimulation ofadenylate cyclase activity which we named 5-HT_(4B). The 5-HT_(4B)receptor was subsequently renamed to the “5-HT₇ receptor” by the“Serotonin Receptor Nomenclature Committee” of the IUPHAR. Thepharmacological properties of this receptor indicated that it wassimilar to a series of functionally defined 5-HT receptors described inthe porcine vena cava (Trevethick et al., 1984), cat saphenous vein,coronary arteries (Cushing and Cohen, 1992), and several vasculardilatory effects (Mylecharane and Phillips, 1989). However, theclassically defined 5-HT₄ receptor remained to be cloned. We now reportthe cloning of the pharmacologically-defined 5-HT₄ receptor which wehave previously called ⁵-HT_(4A) and now designate as the 5-HT₄receptor. This receptor also stimulates adenylate cyclase activity butunlike 5-HT_(4B), is sensitive to a series of benzamide derivativeswhich act as agonists or partial agonists at this subtype. The presenceof this subtype in the brain, particularly in areas such as thehippocampus, indicates a potential role in cognitive enhancement. Inaddition, the 5-HT₄ receptor has been described functionally in theheart, adrenal, bladder, and alimentary canal indicating potential rolesin achalasia, hiatal hernia, esophageal spasm, irritable bowel disease,postoperative ileus, diabetic gastroparesis, emesis and other diseasesof the gastrointestinal tract, as well as in cardiac, urinary, andendocrine function.

SUMMARY OF THE INVENTION

[0004] This invention provides an isolated nucleic acid moleculeencoding a mammalian 5-HT₄ receptor. In a preferred embodiment of thisinvention, the isolated nucleic acid encodes a human 5-HT4 receptor. Inanother embodiment of this invention, the nucleic acid molecule encodinga human 5-HT₄ receptor comprises a plasmid designated pBluescript-hS10(ATCC Accession No. 75392). In another embodiment of this invention anucleic acid molecule encoding a mammalian 5HT4 receptor comprises aplasmid designated pcEXV-S10-87 (ATCC Accession No. 75390). In anotherembodiment of this invention a nucleic acid molecule encoding amammalian 5-HT₄ receptor comprises a plasmid designated pcEXV-S10-95(ATCC Accession No. 75391).

[0005] This invention provides a nucleic acid probe comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding a mammalian 5-HT₄ receptor. Thisinvention also provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence includedwithin the sequence of a nucleic acid molecule encoding a human 5HT-₄receptor.

[0006] This invention provides an antisense oligonucleotide having asequence capable of binding specifically to an mRNA molecule encoding amammalian 5-HT₄ receptor so as to prevent translation of the mRNAmolecule. This invention also provides an antisense oligonucleotidehaving a sequence capable of binding specifically to an mRNA moleculeencoding a human 5-HT₄ receptor so as to prevent translation of the mRNAmolecule.

[0007] This invention provides a monoclonal antibody directed to amammalian 5-HT₄ receptor. This invention also provides a monoclonalantibody directed to a human 5-HT₄ receptor.

[0008] This invention provides a pharmaceutical composition comprisingan amount of a substance effective to alleviate the abnormalitiesresulting from overexpression of a mammalian 5-HT₄ receptor and apharmaceutically acceptable carrier. This invention also provides apharmaceutical composition comprising an amount of a substance effectiveto alleviate abnormalities resulting from underexpression of mammalian5-HT₄ receptor and a pharmaceutically acceptable carrier.

[0009] This invention provides a pharmaceutical composition comprisingan amount of a substance effective to alleviate the abnormalitiesresulting from overexpression of a human 5-HT₄ receptor and apharmaceutically acceptable carrier. This invention also providespharmaceutical composition comprising an amount of a substance effectiveto alleviate abnormalities resulting from underexpression of a human5-HT₄ receptor and a pharmaceutically acceptable carrier.

[0010] This invention provides a transgenic, nonhuman mammal whosegenome comprises DNA encoding a mammalian 5-HT₄ receptor so positionedwithin such genome as to be transcribed into antisense mRNAcomplementary to mRNA encoding the mammalian 5-HT₄ receptor and whenhybridized to mRNA encoding the mammalian 5-HT₄ receptor, thecomplementary mRNA reduces the translation of the mRNA encoding themammalian 5-HT₄ receptor.

[0011] This invention also provides a transgenic, nonhuman mammal whosegenome comprises DNA encoding a human 5-HT₄ so positioned within suchgenome as to be transcribed into antisense mRNA complementary to mRNAencoding the human 5-HT₄ and when hybridized to mRNA encoding the human5-HT₄, the complementary mRNA reduces the translation of the mRNAencoding the human 5-HT₄.

[0012] This invention provides a transgenic, nonhuman mammal whosegenome comprises DNA encoding a mammalian 5-HT₄ receptor so positionedwithin such genome as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the mammalian 5-HT₄ receptor and whenhybridized to mRNA encoding the 5-HT₄ receptor, the antisense mRNAthereby prevents the translation of mRNA encoding the 5-HT₄ receptor.

[0013] This invention also provides a transgenic, nonhuman mammal whosegenome comprises DNA encoding a human 5-HT₄ receptor so positionedwithin such genome as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the human 5-HT₄ receptor and whenhybridized to mRNA encoding the human 5-HT₄ receptor, the antisense mRNAthereby prevents the translation of mRNA encoding the human 5-HT₄receptor.

[0014] This invention also provides a method of determining thephysiological effects of expressing varying levels of a mammalian 5-HT₄receptor which comprises producing a transgenic nonhuman animal whoselevels of mammalian 5-HT₄ receptor expression are varied by use of aninducible promoter which regulates mammalian 5-HT₄ receptor expression.

[0015] This invention also provides a method of determining thephysiological effects of expressing varying levels of a human 5-HT₄receptor which comprises producing a transgenic nonhuman animal whoselevels of human 5-HT₄ receptor expression are varied by use of aninducible promoter which regulates human 5-HT₄ receptor expression.

[0016] This invention further provides a method of determining thephysiological effects of expressing varying levels of mammalian 5-HT₄receptor which comprises producing a panel of transgenic nonhumananimals each expressing a different amount of mammalian 5-HT₄ receptor.

[0017] This invention further provides a method of determining thephysiological effects of expressing varying levels of human 5-HT₄receptor which comprises producing a panel of transgenic nonhumananimals each expressing a different amount of human 5-HT₄ receptor.

[0018] This invention provides a method for determining whether acompound not known to be capable of specifically binding to a human5-HT₄ receptor can specifically bind to the human 5-HT₄ receptor, whichcomprises contacting a mammalian cell comprising a plasmid adapted forexpression in a mammalian cell which plasmid further comprises a DNAwhich expresses a human 5-HT₄ receptor on the cell's surface with thecompound under conditions permitting binding of ligands known to bind toa human 5-HT₄ receptor, detecting the presence of any compound bound tothe human 5-HT₄ receptor, the presence of bound compound indicating thatthe compound is capable of specifically binding to the human 5-HT₄receptor.

[0019] This invention provides a method of screening drugs to identifydrugs which interact with, and specifically bind to, a human 5-HT₄receptor on the surface of a cell, which comprises contacting amammalian cell which comprises a plasmid adapted for expression in amammalian cell which plasmid further comprises DNA which expresses ahuman 5-HT₄ receptor on the cell's surface with a plurality of drugs,determining those drugs which bind to the human 5-HT₄ receptor expressedon the cell surface of the mammalian cell, and thereby identifying drugswhich interact with, and specifically bind to, the human 5-HT₄ receptor.

[0020] This invention provides a method for identifying a compound whichspecifically binds to and activates or blocks the activation of a human5-HT₄ receptor on the surface of a mammalian cell, which comprisescontacting the mammalian cell which comprises a plasmid adapted forexpression in the mammalian cell such plasmid further comprising DNAwhich expresses the human 5-HT₄ receptor on the cell surface of themammalian cell with the compound, determining whether the compoundactivates or blocks the activation of the human 5-HT₄ receptor andthereby identifying the compound as a compound which binds to, andactivates or blocks the activation of the human 5-HT₄ receptor.

[0021] This invention provides a method for diagnosing a predispositionto a disorder associated with the expression of a human 5-HT₄ receptorallele which comprises: a.) obtaining DNA of subjects suffering from thedisorder; b.) performing a restriction digest of the DNA with a panel ofrestriction enzymes; c.) electrophoretically separating the resultingDNA fragments on a sizing gel; d.) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing to DNA encoding a5-HT₄ receptor and labelled with a detectable marker; e.) detectinglabelled bands which have hybridized to the DNA encoding a 5-HT₄receptor labelled with a detectable marker to create a unique bandpattern specific to the DNA of subjects suffering from the disorder; f.)preparing DNA obtained for diagnosis by steps a-e; and g.) comparing theunique band pattern specific to the DNA of subjects suffering from thedisorder from step e and the DNA obtained for diagnosis from step f todetermine whether the patterns are the same or different and to diagnosethereby predisposition to the disorder if the patterns are the same.

BRIEF DESCRIPTION OF FIGURES

[0022]FIG. 1: Nucleotide and corresponding amino acid sequence of theS10-87 cDNA clone. Only partial 5′ and 3′ untranslated sequences areshown.

[0023]FIG. 2: Nucleotide and corresponding amino acid sequence of theS10-95 cDNA clone. Only partial 5′ and 3′ untranslated sequences areshown.

[0024]FIG. 3: Comparison of amino acid sequences between clones S10-87(top row) and S10-95 (bottom row). The overall homology is 96.7%.

[0025]FIG. 4: Comparison of the rat S10 receptor deduced amino acidsequences with those of other serotonin receptors and with the caninehistamine H2 receptor. Solid bars, the seven putative membrane-spanningdomains (TM I-VII). Shading, homologies between the S10 receptors andother receptors. Hp78, 5-HT4B or hp78a receptor (U.S. Ser. No. 971,960,filed, Nov. 3, 1992, copending).

[0026]FIG. 5: Nucleotide and amino acid sequences of the human S10 PCRclone. The numbering is given according to the rat S10-95 clone.

[0027]FIG. 6: Comparison of nucleotide sequences between the human PCRS10 clone and the rat S10 cDNA clone. Top row: human sequence, thenumbering is given according to the rat S10 nucleotide sequence. Thebottom row outlines differences in the rat sequence (overall homology:90.7%).

[0028]FIG. 7: Comparison of deduced amino acid sequences between theHuman S10 PCR clone and the rat S10 cDNA clone. Top row: human S10sequence, the numbering is given according to the rat S10 amino acidsequence. The bottom row outlines differences in the rat sequence(overall homology: 92.3%).

[0029]FIG. 8: Comparison of binding affinities of key compounds at theS10 clone with adenylate cyclase functional responses obtained withmouse collicular neurons. A correlation plot was constructed betweenaffinity constants of drugs for the S10 receptor with those obtained ata pharmacologically defined 5-HT₄ receptor. Binding values for thecorrelation were taken from table 1 and were expressed as the negativelogarithm. Functional data were taken from Dumuis et al. (1988). Thecorrelation coefficient calculated by linear regression was 0.96indicating that the rank order of potency for the compounds was similarin both preparations.

[0030]FIG. 9: Stimulation of cAMP production by 5-HT in transientlytransfected Cos-7 cells expressing the cloned rat 5-HT₄ (CG-7) receptorand antagonism by ICS 205930. cAMP measurements on intact cells were asdescribed under Methods and Materials. Each data point represents themean of triplicates from a single experiment representative of at least2 others. The vertical bars indicate S.E.M. Data are presented aspercent maximum cAMP released by 5-HT (basal CAMP release: 0.020±0.002pmol/ml/10 min; maximum cAMP release: 0.42±0.03 pmol/ml/10 min).

[0031]FIG. 10: Stimulation of cAMP production by 5-HT in transientlytransfected Cos-7 cells expressing the cloned rat 5-HT₄ (CG-8) receptorand antagonism by ICS 205930. cAMP measurements on intact cells were asdescribed under Methods and Materials. Each data point represents themean of triplicates from a single experiment representative of at leasttwo others. The vertical bars indicate S.E.M. Data are presented aspercent maximum cAMP released by 5-HT (basal cAMP release: 0.023±0.004pmol/ml/10 min; maximum CAMP release: 0.57±0.04 pmol/ml/10 min).

[0032]FIG. 11A: Nucleotide sequence of the partial human S10-87 clone.Only partial 3′ untranslated sequences are shown (SEQ. ID NO. 14).

[0033]FIG. 11B: Deduced amino acid sequence encoded by the nucleotidesequence of FIG. 11A of the partial human S10-87 clone (SEQ. ID NO. 15).

[0034]FIG. 12: Comparison of the nucleotide sequences between the human(top row) and the rat S10-87 (bottom row) cDNA clones. The overallidentity is 90.8%.

[0035]FIG. 13: Comparison of the deduced amino acid sequences betweenthe human (top row) and the rat (bottom row) S10-87 receptors. Theoverall identity is 93.9%.

[0036]FIG. 14A: Nucleotide sequence of the full length human S10-95clone (SEQ. ID NO. 7).

[0037]FIG. 14B: Deduced amino acid sequence encoded by the nucleotidesequence of FIG. 14A (SEQ. ID NO. 8).

[0038]FIG. 15: Comparison of the nucleotide sequences between the human(top row) and the rat (bottom row) S10-95 cDNA clones. The overallidentity is 90.7%.

[0039]FIG. 16: Comparison of the deduced amino acid sequences betweenthe human (top row) and the rat (bottom row) S10-95 receptors. Theoverall identity is 93.8%.

[0040]FIG. 17: Comparison of the nucleotide sequences corresponding tothe available coding regions between the two human isoforms (top rowS10-95; bottom row S10-87) of the 5-HT₄ receptor. The overall identityis 92%. 87) FIG. 18: Comparison of the deduced amino acid sequencesbetween the two human isoforms (top row S10-95; bottom row S10-87) ofthe 5-HT₄ receptor. The overall identity is 90%.

[0041]FIG. 19: Inhibition of [³H]GR11380 binding on the cloned rat CG-8receptor by 5-HT, in the absence and presence of Gpp(NH)p (100 μM).Membranes harvested from transient transfectants (COS-7 cells) wereincubated with [³H]IGR113808 (0.2-0.4 nM) for 30 min at 37° C.Nonspecific binding was defined by 50 μM unlabelled 5-HT. Data are froma single experiment. Data were analyzed by computer-assisted nonlinearregression analysis (Accufit; Lundon Software).

DETAILED DESCRIPTION OF THE INVENTION

[0042] This invention provides an isolated nucleic acid moleculeencoding a mammalian 5-HT₄ receptor. This invention further provides anisolated nucleic acid molecule encoding a human 5-HT₄ receptor. As usedherein, the term “isolated nucleic acid molecule” means a non-naturallyoccurring nucleic acid molecule that is, a molecule in a form which doesnot occur in nature. Examples of such an isolated nucleic acid moleculeare an RNA, cDNA, or isolated genomic DNA molecule encoding a mammalian5-HT₄ receptor or a human 5-HT₄ receptor. As used herein, “5-HT₄receptor” means a molecule which, under physiologic conditions, issubstantially specific for the neurotransmitter serotonin, is saturable,of high affinity for serotonin and the activation of which is coupled tothe activation of adenylate cyclase and the “5-HT₄ receptor” is alsosensitive to benzamide derivatives which act as agonists and partialagonists at this receptor subtype. One embodiment of this invention isan isolated nucleic acid molecule encoding a mammalian 5-HT₄ receptor.Such a molecule may have coding sequences substantially the same as thecoding sequences shown in FIGS. 1 and 2 and 5 (SEQ ID NOs. 1, 3 and 5).A preferred embodiment is an isolated nucleic acid molecule encoding ahuman 5-HT₄ receptor. Such a molecule may have a coding sequencesubstantially the same as the coding sequence shown in FIG. 5 (SEQ IDNO. 5). The DNA molecules of FIGS. 1, 2 and 5 (Seq ID NOs. 1, 3 and 5)encode the sequence of mammalian 5-HT₄ receptors. The DNA molecule ofFIG. 5 (Seq ID No. 5) encodes a human 5-HT₄ receptor. This inventionfurther provides isolated DNA molecules encoding mammalian 5-HT₄receptors having the sequence H₂N—Y—X—COOH wherein Y is the amino acidsequence beginning at amino acid 1 and ending at amino acid 359 of FIG.1 (SEQ ID NOs. 1 and 2) and wherein X is an amino acid sequence encodingthe carboxy terminal region of the receptor. The nucleic acid moleculesof FIGS. 1 and 2 (SEQ ID NOs 1-4) encode 5-HT₄ receptors having anidentical sequence Y and differing only in their carboxy terminal regionX beginning at amino acid 360. One means of isolating a nucleic acidmolecule encoding a mammalian 5-HT₄ receptor is to probe a mammaliangenomic library with a natural or artificially designed DNA probe, usingmethods well known in the art. In the preferred embodiment of thisinvention, the mammalian 5-HT₄ receptor is a human protein and thenucleic acid molecule encoding the human 5-HT₄ receptor is isolated fromhuman cDNA. Degenerate oligonucleotide primers derived fromtransmembrane (TM) domains of 5-HT_(1A), 5 -HT_(1C), 5-HT₂ and5-HT_(1Dα/B) receptors are useful for identifying cDNA containing anucleic acid molecule encoding a 5-HT₄ receptor, obtaining a probespecific to a mammalian 5-HT₄ receptor and for isolating a nucleic acidmolecule encoding a mammalian 5-HT₄ receptor.

[0043] DNA and cDNA molecules which encode a mammalian 5-HT₄ receptorare used to obtain complementary genomic DNA, cDNA or RNA from human,mammalian or other animal sources, or to isolate related cDNA or genomicclones by the screening of cDNA or genomic libraries, by methodsdescribed in more detail below. Transcriptional regulatory elements fromthe 5′ untranslated region of the isolated clone, and other stability,processing, transcription, translation, and tissue specificitydetermining regions from the 3′ and 5′ untranslated regions of theisolated gene are thereby obtained.

[0044] This invention provides an isolated nucleic acid molecule whichhas a nucleic acid sequence which differs from the sequence of a nucleicacid molecule encoding a 5-HT₄ receptor at one or more nucleotides andwhich does not encode a protein having 5-HT₄ receptor activity. As usedherein, “5-HT₄ receptor activity” means the capability of receptor tospecifically bind the neurotransmitter, serotonin under physiologicalconditions and the capability of the receptor to activate adenylatecyclase when the receptor is coupled to adenylate cyclase. An example ofa isolated nucleic acid molecule provided by this invention is a nucleicacid molecule which has an in-frame stop codon inserted into the codingsequence such that the transcribed RNA is not translated into protein.

[0045] This invention further provides a cDNA molecule encoding amammalian 5-HT₄ receptor, wherein the cDNA molecule has a codingsequence substantially the same as the coding sequence shown in FIGS. 1,2 and 5 (Seq ID NOs. 1, 3 and 5). This invention provides a cDNAmolecule encoding a human 5-HT₄ receptor, wherein the cDNA molecule hasa coding sequence substantially the same as the coding sequence shown inFIG. 5 (SEQ ID NO. 5). These molecules and their equivalents wereobtained by the means described above.

[0046] This invention also provides an isolated protein which is amammalian 5-HT₄ receptor. In a preferred embodiment of this invention,the protein is a human 5-HT₄ receptor protein having an amino acidsequence substantially similar to the amino acid sequence shown in FIGS.1, 2 and 5 (SEQ ID Nos. 1-6). In another embodiment of this invention,the protein is a murine 5-HT₄ receptor protein having an amino acidsequence substantially similar to the amino acid sequence shown in FIGS.1, 2 and 5 (SEQ ID NOs. 1-6). As used herein, the term “isolatedprotein” is intended to encompass a protein molecule free of othercellular components. One means for obtaining an isolated mammalian 5-HT₄receptor protein is to express DNA encoding the 5-HT₄ receptor in asuitable host, such as a bacterial, yeast, insect, or mammalian cell,using methods well known to those skilled in the art, and recovering thereceptor protein after it has been expressed in such a host, again usingmethods well known in the art. The receptor may also be isolated fromcells which express it, in particular from cells which have beentransfected with the expression vectors described below in more detail.

[0047] This invention provides a vector comprising DNA, RNA, or cDNA,encoding a mammalian 5-HT₄ receptor. This invention further provides avector comprising DNA, RNA, or cDNA, encoding a human 5-HT₄ receptor.Examples of vectors are viruses such as bacteriophages (such as phagelambda), cosmids, plasmids (such as pUCl8, available from Pharmacia,Piscataway, N.J.), and other recombination vectors. Nucleic acidmolecules are inserted into vector genomes by methods well known tothose skilled in the art. Examples of such plasmids are plasmidscomprising DNA having a coding sequence substantially the same as thecoding sequence shown in FIGS. 1, 2 and 5 (SEQ ID NOs. 1, 3 and 5) anddesignated pcEXV-S10-87 (ATCC Accession No. 75390), pcEXV-S10-95 (ATCCAccession No. 75391) and pBLuescript-hS10 (ATCC No. 75392).

[0048] Alternatively, to obtain these vectors, insert and vector DNA canboth be exposed to a restriction enzyme to create complementary ends onboth molecules which base pair with each other and are then ligatedtogether with a ligase. Alternatively, linkers can be ligated to theinsert DNA which correspond to a restriction site in the vector DNA,which is then digested with the restriction enzyme which cuts at thatsite. Other means are also available.

[0049] This invention also provides vectors comprising a DNA or cDNAencoding a mammalian 5-HT₄ receptor and vectors comprising a DNA or cDNAencoding a human 5-HT₄ receptor, adapted for expression in a bacterialcell, a yeast cell, insect cell or a mammalian cell which additionallycomprise the regulatory elements necessary for expression of the DNA orcDNA encoding a mammalian 5-HT₄ receptor or the DNA or cDNA encoding ahuman 5-HT₄ receptor in the bacterial, yeast, insect or mammalian cellsoperatively linked to the DNA or cDNA encoding the 5-HT₄ receptor as topermit expression thereof. DNA or cDNA having coding sequencesubstantially the same as the coding sequence shown in FIGS. 1 and 2(SEQ ID NOs. 1 and 3) may be usefully inserted into these vectors toexpress a mammalian 5-HT₄ receptor. DNA or cDNA having a coding sequencesubstantially the same as the coding sequence shown in FIG. 5 (SEQ IDNO. 5) may be usefully inserted into these vectors to express the human5-HT₄ receptor. Regulatory elements required for expression includepromoter sequences to bind RNA polymerase and transcription initiationsequences for ribosome binding. For example, a bacterial expressionvector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG (Maniatis, et al., 1982). Similarly, a eukaryotic expression vectorincludes a heterologous or homologous promoter for RNA polymerase II, adownstream polyadenylation signal, the start codon AUG, and atermination codon for detachment of the ribosome. Furthermore, an insectexpression vector, such as recombinant Baculovirus, uses the polyhedringene expression signals for expression of the inserted gene in insectcells. Such vectors may be obtained commercially or assembled from thesequences described by methods well known in the art, for example themethods described above for constructing vectors in general. Expressionvectors are useful to produce cells that express receptors. Certain usesfor such cells are described in more detail below.

[0050] In one embodiment of this invention a plasmid is adapted forexpression in a bacterial, yeast, insect, or, in particular, a mammaliancell wherein the plasmid comprises DNA or cDNA encoding a mammalian5-HT₄ receptor or DNA or cDNA encoding a human 5-HT₄ receptor and theregulatory elements necessary for expression of the DNA in thebacterial, yeast, insect, or mammalian cell operatively linked to theDNA or cDNA encoding a mammalian 5-HT₄ receptor or to the DNA or cDNAencoding a human 5-HT₄ receptor as to permit expression thereof.Suitable plasmids may include, but are not limited to plasmids adaptedfor expression in a mammalian cell, e.g., EVJB, EXV-3. An example ofsuch a plasmid adapted for expression in a mammalian cell is a plasmidcomprising cDNA having coding sequences substantially the same as thecoding sequence shown in FIGS. 1, 2 and 5 (SEQ ID NOs. 1, 3 and 5) andthe regulatory elements necessary for expression of the DNA in themammalian cell. These plasmids have been designated pcEXV-S10-87deposited under ATCC Accession No. 75390, pcEXV-S10-95 deposited underATCC Accession No. 75391, and pBluescript-hS10, deposited under ATCCAccession No 75392. Those skilled in the art will readily appreciatethat numerous plasmids adapted for expression in a mammalian cell whichcomprise DNA encoding a mammalian or human 5-HT₄ receptor and theregulatory elements necessary to express such DNA in the mammalian cellmay be constructed utilizing existing plasmids and adapted asappropriate to contain the regulatory elements necessary to express theDNA in the mammalian cell. The plasmids may be constructed by themethods described above for expression vectors and vectors in general,and by other methods well known in the art.

[0051] Deposit discussed supra were made pursuant to, and insatisfaction of, the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

[0052] This invention provides a mammalian cell comprising a DNA or cDNAmolecule encoding a mammalian 5-HT₄ receptor, such as a mammalian cellcomprising a plasmid adapted for expression in a mammalian cell, saidplasmid further comprises DNA or cDNA encoding a mammalian 5-HT₄receptor and the regulatory elements necessary for expression of the DNAor cDNA in the mammalian cell operatively linked to the DNA or cDNAencoding a mammalian 5-HT₄ receptor as to permit expression thereof.This invention provides a mammalian cell comprising a DNA or cDNAmolecule encoding a human 5-HT₄ receptor, such as a mammalian cellcomprising a plasmid adapted for expression in a mammalian cell, saidplasmid further comprises a DNA or cDNA molecule encoding a human 5-HT₄receptor and the regulatory elements necessary for expression of the DNAor cDNA in the mammalian cell operatively linked to the DNA or cDNAencoding a human 5-HT₄ receptor as to permit expression thereof.Numerous mammalian cells may be used as hosts, including, but notlimited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa cells, LM(tk−) cells, Cos-7 cells, etc. Expression plasmids such as thatdescribed supra may be used to transfect mammalian cells by methods wellknown in the art such as calcium phosphate precipitation, or DNA or cDNAencoding a human or mammalian 5-HT₄ receptor may be otherwise introducedinto mammalian cells, e.g., by microinjection, to obtain mammalian cellswhich comprise DNA, e.g., cDNA or a plasmid, encoding a human ormammalian 5-HT₄ receptor.

[0053] This invention provides a nucleic acid probe comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with an unique sequence included within the sequence of anucleic acid molecule encoding a human 5-HT₄ receptor, for example witha coding sequence included within the sequences shown in FIG. 5 (SEQ IDNO. 5). This invention further provides a nucleic acid probe comprisinga nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence included within the sequence ofa nucleic acid molecule encoding a mammalian 5-HT₄ receptor, for examplewith a coding sequence included within the sequences shown in FIG. 1 andFIG. 2 (SEQ ID NOs. 1 and 3) As used herein, the phrase “specificallyhybridizing” means the ability of a nucleic acid molecule to recognize anucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs. As used herein, the phrase “unique sequence” means a nucleicacid molecule sequence specific to only the nucleic acid moleculeencoding a mammalian 5-HT₄ receptor. Nucleic acid probe technology iswell known to those skilled in the art who will readily appreciate thatsuch probes may vary greatly in length and may be labeled with adetectable label, such as a radioisotope or fluorescent dye, tofacilitate detection of the probe. Detection of nucleic acid encoding ahuman 5-HT₄ receptor is useful as a diagnostic test for any diseaseprocess in which levels of expression of the 5-HT₄ receptor are altered.DNA probe molecules are produced by insertion of a DNA molecule whichencodes a 5-HT₄ receptor or fragments thereof into suitable vectors,such as plasmids or bacteriophages, followed by insertion into suitablebacterial host cells and replication and harvesting of the DNA probes,all using methods well known in the art. For example, the DNA may beextracted from a cell lysate using phenol and ethanol, digested withrestriction enzymes corresponding to the insertion sites of the DNA intothe vector (discussed above), electrophoresed, and cut out of theresulting gel. An example of such DNA molecules is shown in FIGS. 1, 2and 5 (SEQ ID NOs. 1, 3, and 5)>. The probes are useful for ‘in situ’hybridization or in order to locate tissues which express this genefamily, or for other hybridization assays for the presence of thesegenes or their mRNA in various biological tissues. In addition,synthesized oligonucleotides (produced by a DNA synthesizer)complementary to the sequence of a DNA molecule which encode a mammalian5-HT₄ receptor or complementary to the sequence of a DNA molecule whichencodes a human 5-HT₄ receptor are useful as probes for these genes, fortheir associated mRNA, or for the isolation of related genes by homologyscreening of genomic or cDNA libraries, or by the use of amplificationtechniques such as the polymerase chain reaction.

[0054] This invention also provides a method of detecting expression ofa human 5-HT₄ receptor on the surface of a cell by detecting thepresence of mRNA coding for a 5-HT₄ receptor. This invention furtherprovides a method of detecting expression of a mammalian 5-HT₄ receptoron the surface of the cell by detecting the presence of mRNA coding fora mammalian 5-HT₄ receptor. These methods comprise obtaining total mRNAfrom the cell using methods well known in the art and contacting themRNA so obtained with a nucleic acid probe as described hereinabove,under hybridizing conditions, detecting the presence of mRNA hybridizedto the probe, and thereby detecting the expression of the receptor bythe cell. Hybridization of probes to target nucleic acid molecules suchas mRNA molecules employs techniques well known in the art. However, inone embodiment of this invention, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing a column which binds the poly-A tails of the mRNA molecules(Maniatis et al., 1982). The mRNA is then exposed to radioactivelylabelled probe on a nitrocellulose membrane, and the probe hybridizes toand thereby labels complementary mRNA sequences. Binding may be detectedby autoradiography or scintillation counting. However, other methods forperforming these steps are well known to those skilled in the art, andthe discussion above is merely an example.

[0055] This invention provides an antisense oligonucleotide having asequence capable of binding specifically with any sequences of an mRNAmolecule which encodes a human 5-HT₄ receptor so as to preventtranslation of the human 5-HT₄ receptor. The antisense oligonucleotidemay have a sequence capable of binding specifically with any sequencesof the cDNA molecule whose sequence is shown in FIG. 5 (SEQ ID NO. 5).This invention also provides an antisense oligonucleotide having asequence capable of binding specifically with any sequences of an mRNAmolecule which encodes a mammalian 5-HT₄ receptor so as to preventtranslation of the mammalian 5-HT₄ receptor. The antisenseoligonucleotide may have a sequence capable of binding specifically withany sequences of the cDNA molecule whose sequence is shown in FIGS. 1and 2 (SEQ ID NOs. 1 and 3). As used herein, the phrase “bindingspecifically” means the ability of an antisense oligonucleotide torecognize a nucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs. A particular example of an antisense oligonucleotide is anantisense oligonucleotide comprising chemical analogues of nucleotides.

[0056] This invention also provides a pharmaceutical compositioncomprising an effective amount of the oligonucleotide described aboveeffective to reduce expression of a human 5-HT₄ receptor by passingthrough a cell membrane and binding specifically with mRNA encoding the5-HT₄ receptor in the cell so as to prevent its translation and apharmaceutically acceptable hydrophobic carrier capable of passingthrough a cell membrane. This invention further provides apharmaceutical composition comprising an effective amount of theoligonucleotide described above effective to reduce expression of amammalian 5-HT₄ receptor by passing through a cell membrane and bindingspecifically with mRNA encoding a mammalian 5-HT₄ receptor in the cellso as to prevent its translation and a pharmaceutically acceptablehydrophobic carrier capable of passing through a cell membrane. As usedherein, the term “pharmaceutically acceptable carrier” encompasses anyof the standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents. The oligonucleotide maybe coupled to a substance which inactivates mRNA, such as a ribozyme.The pharmaceutically acceptable hydrophobic carrier capable of passingthrough cell membranes may also comprise a structure which binds to atransporter specific for a selected cell type and is thereby taken up bycells of the selected cell type. The structure may be part of a proteinknown to bind a cell-type specific transporter, for example an insulinmolecule, which would target pancreatic cells. DNA molecules having acoding sequence substantially the same as the coding sequences shown inFIG. 5 (SEQ ID No. 5) may be used as the oligonucleotides of thepharmaceutical composition.

[0057] This invention also provides a pharmaceutical compositioncomprising an effective amount of the oligonucleotide described aboveeffective to reduce expression of a mammalian 5-HT₄ receptor by passingthrough a cell membrane and binding specifically with mRNA encoding the5-HT₄ receptor in the cell so as to prevent its translation and apharmaceutically acceptable hydrophobic carrier capable of passingthrough a cell membrane. DNA molecules having a coding sequencesubstantially the same as the coding sequences shown in FIGS. 1 and 2(SEQ ID NOs. 1 and 3) may be used as the oligonucleotides of thepharmaceutical composition.

[0058] This invention provides a method of treating abnormalities whichare alleviated by reduction of expression of 5-HT₄ receptor. This methodcomprises administering to a subject an effective amount of thepharmaceutical composition described above effective to reduceexpression of the 5-HT₄ receptor by the subject. This invention furtherprovides a method of treating an abnormal condition related to 5-HT₄receptor activity which comprises administering to a subject an amountof the pharmaceutical composition described above effective to reduceexpression of the 5-HT₄ receptor by the subject. Examples of suchabnormal conditions are irritable bowel disease, postoperative ileus,diabetic gastroparesis, emesis, achalasia, hiatal hernia, esophagealspasm and other diseases of the gastrointestinal tract, as well as incardiac, urinary, and endocrine function.

[0059] Antisense oligonucleotide drugs inhibit translation of mRNAencoding 5-HT₄ receptor. Synthetic antisense oligonucleotides, or otherantisense chemical structures are designed to bind to mRNA encoding the5-HT₄ receptor and inhibit translation of mRNA and are useful as drugsto inhibit expression of 5-HT₄ receptor genes in patients. Thisinvention provides a means to therapeutically alter levels of expressionof a human or mammalian 5-HT₄ receptor by the use of a syntheticantisense oligonucleotide drug (SAOD) which inhibits translation of mRNAencoding the 5-HT₄ receptor. Synthetic antisense oligonucleotides, orother antisense chemical structures designed to recognize andselectively bind to mRNA, are constructed to be complementary toportions of the nucleotide sequence shown in FIGS. 1, 2 and 5 (SEQ IDNOs. 1, 3 and 5) of DNA, RNA or of chemically modified, artificialnucleic acids. The SAOD is designed to be stable in the blood stream foradministration to patients by injection, or in laboratory cell cultureconditions, for administration to cells removed from the patient. TheSAOD is designed to be capable of passing through cell membranes inorder to enter the cytoplasm of the cell by virtue of physical andchemical properties of the SAOD which render it capable of passingthrough cell membranes (e.g., by designing small, hydrophobic SAODchemical structures) or by virtue of specific transport systems in thecell which recognize and transport the SAOD into the cell. In addition,the SAOD can be designed for administration only to certain selectedcell populations by targeting the SAOD to be recognized by specificcellular uptake mechanisms which bind and take up the SAOD only withincertain selected cell populations. For example, the SAOD may be designedto bind to transporter found only in a certain cell type, as discussedabove. The SAOD is also designed to recognize and selectively bind tothe target mRNA sequence, which may correspond to a sequence containedwithin the sequence shown in FIGS. 1, 2 and 5 (SEQ ID NOs. 1, 3, and 5)by virtue of complementary base pairing to the mRNA. Finally, the SAODis designed to inactivate the target mRNA sequence by any of threemechanisms: 1) by binding to the target mRNA and thus inducingdegradation of the mRNA by intrinsic cellular mechanisms such as RNAse Idigestion, 2) by inhibiting translation of the mRNA target byinterfering with the binding of translation-regulating factors or ofribosomes, or 3) by inclusion of other chemical structures, such asribozyme sequences or reactive chemical groups, which either degrade orchemically modify the target mRNA. Synthetic antisense oligonucleotidedrugs have been shown to be capable of the properties described abovewhen directed against mRNA targets (Cohen, J. S., 1989; Weintraub, H.M., 1990). In addition, coupling of ribozymes to antisenseoligonucleotides is a promising strategy for inactivating target mRNA(N. Sarver et al., 1990). An SAOD serves as an effective therapeuticagent if it is designed to be administered to a patient by injection, orif the patient's target cells are removed, treated with the SAOD in thelaboratory, and replaced in the patient. In this manner, an SAOD servesas a therapy to reduce 5-HT₄ receptor expression in particular targetcells of a patient, in any clinical condition which may benefit fromreduced expression of 5-HT₄ receptor.

[0060] This invention provides an antibody directed to the human 5-HT₄receptor. This invention also provides an antibody directed to themammalian 5-HT₄ receptor. This antibody may comprise, for example, amonoclonal antibody directed to an epitope of a human 5-HT₄ receptorpresent on the surface of a cell, the epitope having an amino acidsequence substantially the same as an amino acid sequence for a cellsurface epitope of the human 5-HT₄ receptor included in the amino acidsequence shown in FIG. 5. Amino acid sequences may be analyzed bymethods well known to those skilled in the art to determine whether theyproduce hydrophobic or hydrophilic regions in the proteins which theybuild. In the case of cell membrane proteins, hydrophobic regions arewell known to form the part of the protein that is inserted into thelipid bilayer which forms the cell membrane, while hydrophilic regionsare located on the cell surface, in an aqueous environment. Thereforeantibodies to the hydrophilic amino acid sequences shown in FIG. 5 willbind to a surface epitope of a 5-HT₄ receptor as described. Antibodiesdirected to a human or mammalian 5-HT₄ receptor may be serum-derived ormonoclonal and are prepared using methods well known in the art. Forexample, monoclonal antibodies are prepared using hybridoma technologyby fusing antibody producing B cells from immunized animals with myelomacells and selecting the resulting hybridoma cell line producing thedesired antibody. Cells such as NIH3T3 cells or LM (tk⁻) cells may beused as immunogens to raise such an antibody. Alternatively, syntheticpeptides may be prepared using commercially available machines and theamino acid sequence shown in FIGS. 1, 2, and 5 (SEQ ID NOs. 1-6). As astill further alternative, DNA, such as a cDNA or a fragment thereof,may be cloned and expressed and the resulting polypeptide recovered andused as an immunogen. These antibodies are useful to detect the presenceof 5-HT₄ receptor encoded by the isolated DNA, or to inhibit thefunction of the 5-HT₄ receptor in living animals, in humans, or inbiological tissues or fluids isolated from animals or humans.

[0061] This invention also provides a pharmaceutical composition whichcomprises an effective amount of an antibody directed to an epitope ofthe human 5-HT₄ receptor, effective to block binding of naturallyoccurring substrates to the 5-HT₄ receptor, and a pharmaceuticallyacceptable carrier. A monoclonal antibody directed to an epitope of ahuman 5-HT₄ receptor present on the surface of a cell which has an aminoacid sequence substantially the same as an amino acid sequence for acell surface epitope of the human 5-HT₄ receptor included in the aminoacid sequence shown in FIG. 5 (SEQ ID NOs. 5 and 6) is useful for thispurpose.

[0062] This invention also provides a pharmaceutical composition whichcomprises an effective amount of an antibody directed to an epitope of amammalian 5-HT₄ receptor, effective to block binding of naturallyoccurring substrates to the 5-HT₄ receptor, and a pharmaceuticallyacceptable carrier. A monoclonal antibody directed to an epitope of amammalian 5-HT₄ receptor present on the surface of a cell which has anamino acid sequence substantially the same as an amino acid sequence fora cell surface epitope of a mammalian 5-HT₄ receptor included in theamino acid sequence shown in FIGS. 1 and 2 (SEQ ID NOs. 1-4) is usefulfor this purpose.

[0063] This invention also provides a method of treating abnormalitiesin a subject which are alleviated by reduction of expression of a humanor mammalian 5-HT₄ receptor which comprises administering to the subjectan effective amount of the pharmaceutical composition described aboveeffective to block binding of naturally occurring substrates to thereceptor and thereby alleviate abnormalities resulting fromoverexpression of a human or mammalian 5-HT₄ receptor. Binding of theantibody to the receptor prevents the receptor from functioning, therebyneutralizing the effects of overexpression. The monoclonal antibodiesdescribed above are useful for this purpose. This invention additionallyprovides a method of treating an abnormal condition related to an excessof 5-HT₄ receptor activity which comprises administering to a subject anamount of the pharmaceutical composition described above effective toblock binding of naturally occurring substrates to the 5-HT₄ receptorand thereby alleviate the abnormal condition. Some examples of abnormalconditions associated with excess 5-HT₄ receptor activity are irritablebowel disease, postoperative ileus, diabetic gastroparesis, emesis,achalasia, hiatal hernia, esophageal spasm and other diseases of thegastrointestinal tract, as well as in cardiac, urinary, and endocrinefunction.

[0064] This invention provides methods of detecting the presence of a5-HT₄ receptor on the surface of a cell which comprises contacting thecell with an antibody directed to the 5-HT₄ receptor, under conditionspermitting binding of the antibody to the receptor, detecting thepresence of the antibody bound to the cell, and thereby the presence ofthe 5-HT₄ receptor on the surface of the cell. Such methods are usefulfor determining whether a given cell is defective in expression of 5-HT₄receptors. Bound antibodies are detected by methods well known in theart, for example by binding fluorescent markers to the antibodies andexamining the cell sample under a fluorescence microscope to detectfluorescence on a cell indicative of antibody binding. The monoclonalantibodies described above are useful for this purpose.

[0065] This invention provides a transgenic nonhuman mammal expressingDNA encoding a human 5-HT₄ receptor and a transgenic nonhuman mammalexpressing DNA encoding a mammalian 5-HT₄ receptor. This invention alsoprovides a transgenic nonhuman mammal expressing DNA encoding a human ormammalian 5-HT₄ receptor so mutated as to be incapable of normalreceptor activity, and not expressing native 5-HT₄ receptor. Thisinvention further provides a transgenic nonhuman mammal whose genomecomprises DNA encoding a human 5-HT₄ receptor so placed as to betranscribed into antisense mRNA which is complementary to mRNA encodinga human 5-HT₄ receptor and which hybridizes to mRNA encoding a 5-HT₄receptor thereby reducing its translation and a transgenic nonhumanmammal whose genome comprises DNA encoding a mammalian 5-HT₄ receptor soplaced as to be transcribed into antisense mRNA which is complementaryto mRNA encoding a mammalian 5-HT₄ receptor and which hybridizes to mRNAencoding a mammalian 5-HT₄ receptor thereby reducing its translation.The DNA may additionally comprise an inducible promoter or additionallycomprise tissue specific regulatory elements, so that expression can beinduced, or restricted to specific cell types. Examples of DNA are DNAor cDNA molecules having a coding sequence substantially the same as thecoding sequences shown in FIGS. 1,2 and 5 (SEQ ID NOs. 1, 3, and 5). Anexample of a transgenic animal is a transgenic mouse. Examples of tissuespecificity-determining regions are the metallothionein promotor (Low etal., 1986) and the L7 promotor (Oberdick et al., 1990).

[0066] Animal model systems which elucidate the physiological andbehavioral roles of mammalian receptors are produced by creatingtransgenic animals in which the expression of a receptor is eitherincreased or decreased, or the amino acid sequence of the expressedreceptor protein is altered, by a variety of techniques. Examples ofthese techniques include, but are not limited to: 1) Insertion of normalor mutant versions of DNA encoding a human 5-HT₄ receptor or homologousanimal versions of this gene, by microinjection, retroviral infection orother means well known to those skilled in the art, into appropriatefertilized embryos in order to produce a transgenic animal (Hogan etal., 1986) or, 2) Homologous recombination (Capecchi M. R., 1989; ZimmerA, and Gruss, P., 1989) of mutant or normal, human or animal versions ofthese genes with the native gene locus in transgenic animals to alterthe regulation of expression or the structure of the receptor. Thetechnique of homologous recombination is well known in the art. Itreplaces the native gene with the inserted gene and so is useful forproducing an animal that cannot express native receptor but doesexpress, for example, an inserted mutant receptor, which has replacedthe native receptor in the animal's genome by recombination, resultingin underexpression of the receptor. Microinjection adds genes to thegenome, but does not remove them, and so is useful for producing ananimal which expresses its own and added receptors, resulting inoverexpression of the receptor.

[0067] One means available for producing a transgenic animal, with amouse as an example, is as follows: Female mice are mated, and theresulting fertilized eggs are dissected out of their oviducts. The eggsare stored in an appropriate medium such as M2 medium (Hogan, B. et al.1986). DNA or cDNA encoding a receptor is purified from a vector (suchas plasmids pcEXV-S10-87, pcEXV-S10-95 and pBluescript-hS10 describedabove) by methods well known in the art. Inducible promoters may befused with the coding region of the DNA to provide an experimental meansto regulate expression of the trans-gene. Alternatively or in addition,tissue specific regulatory elements may be fused with the coding regionto permit tissue-specific expression of the trans-gene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipet puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant), where it proceeds to theuterus, implants, and develops to term. As noted above, microinjectionis not the only method for inserting DNA into the egg cell, and is usedhere only for exemplary purposes.

[0068] Since the normal action of receptor-specific drugs is to activateor to inhibit the receptor, the transgenic animal model systemsdescribed above are useful for testing the biological activity of drugsdirected against the receptors even before such drugs become available.These animal model systems are useful for predicting or evaluatingpossible therapeutic applications of drugs which activate or inhibitreceptors by inducing or inhibiting expression of the native ortrans-gene and thus increasing or decreasing expression of normal ormutant receptors in the living animal. Thus, a model system is producedin which the biological activity of drugs directed against the receptorsare evaluated before such drugs become available. The transgenic animalswhich over or under produce the receptor indicate by their physiologicalstate whether over or under production of the receptor istherapeutically useful. It is therefore useful to evaluate drug actionbased on the transgenic model system. One use is based on the fact thatit is well known in the art that a drug such as an antidepressant actsby blocking neurotransmitter uptake, and thereby increases the amount ofneurotransmitter in the synaptic cleft. The physiological result of thisaction is to stimulate the production of less receptor by the affectedcells, leading eventually to underexpression. Therefore, an animal whichunderexpresses receptor is useful as a test system to investigatewhether the actions of such drugs which result in under expression arein fact therapeutic. Another use is that if overexpression is found tolead to abnormalities, then a drug which down-regulates or acts as anantagonist to the receptor is indicated as worth developing, and if apromising therapeutic application is uncovered by these animal modelsystems, activation or inhibition of the 5-HT₄ receptor is achievedtherapeutically either by producing agonist or antagonist drugs directedagainst the 5-HT₄ receptor or by any method which increases or decreasesthe expression of this receptor in man.

[0069] Further provided by this invention is a method of determining thephysiological effects of expressing varying levels of human or mammalian5-HT₄ receptors which comprises producing a transgenic nonhuman animalwhose levels of human or mammalian 5-HT₄ receptor expression are variedby use of an inducible promoter which regulates receptor expression.This invention also provides a method of determining the physiologicaleffects of expressing varying levels of human or mammalian 5-HT₄receptor which comprises producing a panel of transgenic nonhumananimals each expressing a different amount of human or mammalian 5-HT₄receptor. Such animals may be produced by introducing different amountsof DNA encoding a human or mammalian 5-HT₄ receptor into the oocytesfrom which the transgenic animals are developed.

[0070] This invention also provides a method for identifying a substancecapable of alleviating abnormalities resulting from overexpression of ahuman or mammalian 5-HT₄ receptor comprising administering the substanceto a transgenic nonhuman mammal expressing at least one artificiallyintroduced DNA molecule encoding a human or mammalian 5-HT₄ receptor anddetermining whether the substance alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofoverexpression of a human or mammalian 5-HT₄ receptor. As used herein,the term “substance” means a compound or composition which may benatural, synthetic, or a product derived from screening. Examples of DNAmolecules are DNA or cDNA molecules having a coding sequencesubstantially the same as the coding sequences shown in FIGS. 1, 2, and5 (SEQ ID NOs. 1, 3, and 5).

[0071] This invention provides a pharmaceutical composition comprisingan amount of the substance described supra effective to alleviate theabnormalities resulting from overexpression of 5-HT₄ receptor and apharmaceutically acceptable carrier.

[0072] This invention further provides a method for treating theabnormalities resulting from overexpression of a human or mammalian5-HT₄ receptor which comprises administering to a subject an amount ofthe pharmaceutical composition described above effective to alleviatethe abnormalities resulting from overexpression of a human or mammalian5-HT₄ receptor.

[0073] This invention provides a method for identifying a substancecapable of alleviating the abnormalities resulting from underexpressionof a human or mammalian 5-HT₄ receptor comprising administering thesubstance to the transgenic nonhuman mammal described above whichexpresses only nonfunctional human or mammalian 5-HT₄ receptor anddetermining whether the substance alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofunderexpression of a human or mammalian 5-HT₄ receptor.

[0074] This invention also provides a pharmaceutical compositioncomprising an amount of a substance effective to alleviate abnormalitiesresulting from underexpression of a human or mammalian 5-HT₄ receptorand a pharmaceutically acceptable carrier.

[0075] This invention further provides a method for treating theabnormalities resulting from underexpression of a human or mammalian5-HT₄ receptor which comprises administering to a subject an amount ofthe pharmaceutical composition described above effective to alleviatethe abnormalities resulting from underexpression of a human or mammalian5-HT₄ receptor.

[0076] This invention provides a method for diagnosing a predispositionto a disorder associated with the expression of a human or mammalian5-HT₄ receptor allele which comprises: a) obtaining DNA of subjectssuffering from the disorder; b) performing a restriction digest of theDNA with a panel of restriction enzymes; c) electrophoreticallyseparating the resulting DNA fragments on a sizing gel; d) contactingthe resulting gel with a nucleic acid probe capable of specificallyhybridizing to DNA encoding a human or mammalian 5-HT₄ receptor andlabelled with a detectable marker; e) detecting labelled bands whichhave hybridized to the DNA encoding a human or mammalian 5-HT₄ receptorlabelled with a detectable marker to create a unique band patternspecific to the DNA of subjects suffering from the disorder; f)preparing DNA obtained for diagnosis by steps a-e; and g) comparing theunique band pattern specific to the DNA of subjects suffering from thedisorder from step e and the DNA obtained for diagnosis from step f todetermine whether the patterns are the same or different and thereby todiagnose predisposition to the disorder if the patterns are the same.This method may also be used to diagnose a disorder associated with theexpression of a specific human 5-HT₄ receptor allele or mammalian 5-HT₄receptor allele.

[0077] This invention provides a method of preparing the isolated 5-HT₄receptor which comprises inducing cells to express receptor, recoveringthe receptor from the resulting cells, and purifying the receptor sorecovered. An example of a 5-HT₄ receptor is an isolated protein havingsubstantially the same amino acid sequence as the amino acid sequenceshown in FIG. 5. For example, cells can be induced to express receptorsby exposure to substances such as hormones. The cells can then behomogenized and the receptor isolated from the homogenate using anaffinity column comprising, for example serotonin or another substancewhich is known to bind to the 5-HT₄ receptor. The resulting fractionscan then be purified by contacting them with an ion exchange column, anddetermining which fraction contains 5-HT₄ receptor activity or bindsanti-receptor antibodies.

[0078] This invention provides a method of preparing an isolated human5-HT₄ receptor which comprises inserting nucleic acid encoding the human5-HT₄ receptor in a suitable vector, inserting the resulting vector in asuitable host cell, recovering the receptor produced by the resultingcell, and purifying the receptor so recovered. An example of an isolatedhuman 5-HT₄ receptor is an isolated protein having substantially thesame amino acid sequence as the amino acid sequence shown in FIG. 5 (SEQID NOs. 5 and 6). This invention provides a method of preparing anisolated mammalian 5-HT₄ receptor which comprises inserting nucleic acidencoding the mammalian 5-HT₄ receptor in a suitable vector, insertingthe resulting vector in a suitable host cell, recovering the receptorproduced by the resulting cell, and purifying the receptor so recovered.An example of an isolated mammalian 5-HT₄ receptor is an isolatedprotein having substantially the same amino acid sequence as the aminoacid sequence shown in FIGS. 1 and 2 (SEQ ID NOs. 1-2 and Seq I. D. Nos.3-4, respectively). These methods for preparing 5-HT₄ receptor usesrecombinant DNA technology methods well known in the art. For example,isolated nucleic acid encoding 5-HT₄ receptor is inserted in a suitablevector, such as an expression vector. A suitable host cell, such as abacterial cell, insect cell, or a eukaryotic cell such as a yeast cell,is transfected with the vector. 5-HT₄ receptor is isolated from theculture medium by affinity purification or by chromatography or othermethods well known in the art.

[0079] This invention provides a method for determining whether acompound not known to be capable of specifically binding to a human5-HT₄ receptor can specifically bind to the human 5-HT₄ receptor, whichcomprises contacting a mammalian cell comprising a plasmid adapted forexpression in a mammalian cell which plasmid further comprises a DNAwhich expresses a human 5-HT₄ receptor on the cell's surface with thecompound under conditions permitting binding of ligands known to bind toa human 5-HT₄ receptor, detecting the presence of any compound bound tothe human 5-HT₄ receptor, the presence of bound compound indicating thatthe compound is capable of specifically binding to the human 5-HT₄receptor.

[0080] This invention provides a method for determining whether acompound not known to be capable of specifically binding to a mammalian5-HT₄ receptor can specifically bind to the mammalian 5-HT₄ receptor,which comprises contacting a mammalian cell comprising a plasmid adaptedfor expression in a mammalian cell which plasmid further comprises a DNAwhich expresses a mammalian 5-HT₄ receptor on the cell's surface withthe compound under conditions permitting binding of ligands known tobind to a mammalian 5-HT₄ receptor, detecting the presence of anycompound bound to the human 5-HT₄ receptor, the presence of boundcompound indicating that the compound is capable of specifically bindingto the mammalian 5-HT₄ receptor.

[0081] This invention provides a method for identifying a compound whichis not known to be capable of binding to a human 5-HT₄ receptor canfunctionally activate the human 5-HT₄ receptor on the surface of amammalian cell or prevent a ligand which does so, which comprisescontacting the mammalian cell which cell comprises a plasmid adapted forexpression in the mammalian cell such plasmid further comprising DNAwhich expresses the human 5-HT₄ receptor on the surface of the mammaliancell with the compound, determining whether the compound activates thehuman 5-HT₄ receptor or prevents a ligand which does so, and therebyidentifying the compound as a compound which is binds to andfunctionally activates the human 5-HT₄ receptor or prevents thefunctional activation of the human 5-HT₄ receptor by a ligand which doesso. The DNA in the cell may have a coding sequence substantially thesame as the coding sequence shown in FIG. 5 ( SEQ ID No. 5).

[0082] This invention provides a method for identifying a compound whichis not known to be capable of binding to a mammalian 5-HT₄ receptor canfunctionally activate the mammalian 5-HT₄ receptor on the surface of amammalian cell or prevent a ligand which does so, which comprisescontacting the mammalian cell which cell comprises a plasmid adapted forexpression in the mammalian cell such plasmid further comprising DNAwhich expresses the mammalian 5-HT₄ receptor on the surface of themammalian cell with the compound, determining whether the compoundactivates the mammalian 5-HT₄ receptor or prevents a ligand which doesso, and thereby identifying the compound as a compound which is binds toand functionally activates the mammalian 5-HT₄ receptor or prevents thefunctional activation of the mammalian 5-HT₄ receptor by a ligand whichdoes so. The DNA in the cell may have a coding sequence substantiallythe same as the coding sequence shown in FIGS. 1 and 2 (SEQ ID NOs. 1and 3).

[0083] The activation or blockade of the functional response is detectedby means of a bioassay from the mammalian cell such as a secondmessenger response, and thereby determining whether the compoundactivates or prevents the activation of the 5-HT₄ receptor functionaloutput. Preferably, the mammalian cell is nonneuronal in origin. Anexample of a nonneuronal mammalian cell is an LM (tk−) cell. Anotherexample of a non-neuronal mammalian cell to be used for functionalassays is a murine fibroblast cell line, specifically the NIH3T3 cell.The preferred method for determining whether a compound is capable ofbinding to the 5-HT₄ receptor comprises contacting a transfectednonneuronal mammalian cell (i.e. a cell that does not naturally expressany type of 5-HT or G-protein coupled receptor, thus will only expresssuch a receptor if it is transfected into the cell) expressing a 5-HT₄receptor on its surface, or contacting a membrane preparation derivedfrom such a transfected cell, with the compound under conditions whichare known to prevail, and thus to be associated with, in vivo binding ofligands to a 5-HT₄ receptor, detecting the presence of any of thecompound being tested bound to the 5-HT₄ receptor on the surface of thecell, and thereby determining whether the compound binds to, andactivates or prevents the activation of the 5-HT₄ receptor. Thisresponse system is obtained by transfection of isolated DNA into asuitable host cell containing the desired second messenger system suchas phosphoinositide hydrolysis, adenylate cyclase, guanylate cyclase orion channels. Such a host system is isolated from pre-existing celllines, or can be generated by inserting appropriate components of secondmessenger systems into existing cell lines. Such a transfection systemprovides a complete response system for investigation or assay of theactivity of human 5-HT₄ receptor with compounds as described above.

[0084] Transfection systems are useful as living cell cultures forcompetitive binding assays between known or candidate drugs and ligandswhich bind to the receptor and which are labeled by radioactive,spectroscopic or other reagents. Membrane preparations containing thereceptor isolated from transfected cells are also useful for thesecompetitive binding assays. Functional assays of second messengersystems or their sequelae in transfection systems act as assays forbinding affinity and efficacy in the activation of receptor function. Atransfection system constitutes a “drug discovery system” useful for theidentification of natural or synthetic compounds with potential for drugdevelopment that can be further modified or used directly as therapeuticcompounds to activate or inhibit the natural functions of the human5-HT₄ receptor. The transfection system is also useful for determiningthe affinity and efficacy of known drugs at human 5-HT₄ receptor sites.

[0085] This invention also provides a method of screening compounds toidentify drugs which interact with, and specifically bind to, a human5-HT₄ receptor on the surface of a cell, which comprises contacting amammalian cell which comprises a plasmid adapted for expression in amammalian cell which plasmid further comprises DNA which expresses ahuman 5-HT₄ receptor on the cell's surface with a plurality ofcompounds, determining those compounds which bind to the human 5-HT₄receptor expressed on the cell surface of the mammalian cell, andthereby identifying compounds which interact with, and specifically bindto, the human 5-HT₄ receptor. The DNA in the cell may have a codingsequence substantially the same as the coding sequence shown in FIG. 5(SEQ ID NO. 5). This invention also provides a method of screeningcompounds to identify drugs which interact with, and specifically bindto, a mammalian 5-HT₄ receptor on the surface of a cell, which comprisescontacting a mammalian cell which comprises a plasmid adapted forexpression in a mammalian cell which plasmid further comprises DNA whichexpresses a mammalian 5-HT₄ receptor on the cell's surface with aplurality of compounds, determining those compounds which bind to themammalian 5-HT₄ receptor expressed on the cell surface of the mammaliancell, and thereby identifying compounds which interact with, andspecifically bind to, the mammalian 5-HT₄ receptor. The DNA in the cellmay have a coding sequence substantially the same as the coding sequenceshown in FIGS. 1 and 3 (SEQ ID NOs. 1 and 2). Various methods ofdetection may be employed. The compounds may be “labeled” by associationwith a detectable marker substance (e.g., radiolabel or a non-isotopiclabel such as biotin). Preferably, the mammalian cell is nonneuronal inorigin. An example of a nonneuronal mammalian cell is a Cos-7 cell. Drugcandidates are identified by choosing chemical compounds which bind withhigh affinity to the expressed 5-HT₄ receptor protein in transfectedcells, using radioligand binding methods well known in the art, examplesof which are shown in the binding assays described herein. Drugcandidates are also screened for selectivity by identifying compoundswhich bind with high affinity to one particular receptor but do not bindwith high affinity to any other receptor subtypes or to any other knownreceptor. Because selective, high affinity compounds interact primarilywith the target 5-HT₄ receptor site after administration to the patient,the chances of producing a drug with unwanted side effects are minimizedby this approach.

[0086] This invention provides a pharmaceutical composition comprising adrug identified by the method described above and a pharmaceuticallyacceptable carrier. As used herein, the term “pharmaceuticallyacceptable carrier” encompasses any of the standard pharmaceuticalcarriers, such as a phosphate buffered saline solution, water, andemulsions, such as an oil/water or water/oil emulsion, and various typesof wetting agents. Once the candidate drug has been shown to beadequately bioavailable following a particular route of administration,for example orally or by injection (adequate therapeutic concentrationsmust be maintained at the site of action for an adequate period to gainthe desired therapeutic benefit), and has been shown to be non-toxic andtherapeutically effective in appropriate disease models, the drug may beadministered to patients by that route of administration determined tomake the drug bioavailable, in an appropriate solid or solutionformulation, to gain the desired therapeutic benefit.

[0087] Applicants have identified a novel 5-HT₄ receptor subtypeprotein, designated 5-HT₄ and have described methods for theidentification of pharmacological compounds for therapeutic treatments.Pharmacological compounds which are directed against specific receptorsubtypes provide effective new therapies with minimal side effects.

[0088] Elucidation of the molecular structures of the neuronal serotoninreceptors is an important step in the understanding of serotonergicneurotransmission. This disclosure reports the isolation and amino acidsequence of a novel cDNA which encodes a human 5-HT₄ receptor. Thisdisclosure reports the isolation, amino acid sequence, and functionalexpression of a two novel cDNAs which encode mammalian 5-HT₄ receptors.The identification of 5-HT₄ receptor subtypes play a pivotal role inelucidating the molecular mechanisms underlying serotonergictransmission, and should also aid in the development of noveltherapeutic agents.

[0089] A complementary DNA clone (designated pBluescript-hS10) encodinga serotonin receptor subtype, 5-HT₄, has been isolated from human brain,human heart and human retina. Additionally, two complementary DNA clonesencoding the serotonin 5-HT₄ receptor subtype have been isolated frommammalian brain and their functional properties have been examined inmammalian cells. Analysis of 5-HT₄ structure and function provides amodel for the development of drugs useful for the treatment ofgastrointestinal conditions including irritable bowel disease,postoperative ileus, diabetic gastroparesis, emesis, achalasia, hiatalhernia, and esophageal spasm. In addition, 5-HT₄ receptors have beendescribed functionally in the heart (Kaumann, 1992), adrenal (Lefebvreet al., 1992), and bladder (Corsi et al., 1991) indicating possibleroles in cardiac rate and force of contraction, endocrine control ofcortisol secretion, and urinary incontinence or spasticity. 5-HT₄receptors have also been described in the brain, particularly in areassuch as the hippocampus, in which we have localized the gene encoding5-HT₄ receptors (S10-95), indicating a potential role in cognitiveenhancement (Bockaert et al., 1992).

[0090] This invention identifies a mammalian serotonin receptor, itsamino acid sequence, and its mammalian gene, the activation of which iscoupled to activation of adenylate cyclase. The information andexperimental tools provided by this discovery are useful to generate newtherapeutic agents, and new therapeutic or diagnostic assays for thisreceptor protein, its associated mRNA molecule or its associated genomicDNA. The information and experimental tools provided by this discoverywill be useful to generate new therapeutic agents, and new therapeuticor diagnostic assays for this new serotonin receptor subtype, itsassociated mRNA molecule, or its associated genoric DNA.

[0091] Specifically, this invention relates to the isolation of humancDNA clone and mammalian cDNA clones encoding a new serotonin receptor,designated 5-HT₄. In addition, the mammalian 5-HT₄ receptors have beenexpressed in COS-7 cells by transfecting the cells with the plasmidspcEXV-S10-87 and pcEXV-S10-95. The pharmacological binding properties ofthe encoded 5-HT₄ receptor have been determined, and the bindingproperties classify this receptor as a novel serotonin receptor.Mammalian cell lines expressing the mammalian 5-HT₄ receptor on the cellsurface have been constructed, thus establishing the first well-defined,cultured cell lines with which to study the novel 5-HT₄ receptor.

[0092] The invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative, and are not meant to limit the invention as describedherein, which is defined by the claims which follow thereafter.

[0093] Methods and Materials

[0094] PCR Amplification: The third (III) and fifth (V) transmembrane(TM) domains of the following receptors were aligned and used tosynthesize a pair of degenerate primers: 5-HT_(1A), 5-HT_(1C), 5-HT₂ andthe 5-HT_(1Dα/β) receptors. Primers 3.17 and 5.5([5′-TGGAATTCTG(C/T)G(C/T)IAT(A/C/T)(G/T)CICTGGA(C/T)(A/C) G(C/G)TA-3′](SEQ ID No. 9),[5′-CATIA(G/C/A)I(G/A)IIA(G/A)IGG(T/G/A/)AT(G/A)(T/A)A(G/A)AAIGC-3′])(SEQ ID No. 10) were used to amplify 5 μg of poly (A+) RNA from ratbrain that was reverse transcribed by avian myeloblastosis virus reversetranscriptase (AMV). PCR was performed on single-stranded cDNA under thefollowing conditions: 94° C. for 1 min, 50° C. for 2 min and 72° C. for3 min for 40 cycles. Following PCR, 90 μl of the reaction wasphenol:chloroform extracted and precipitated; 10μl was visualized on agel using ethidium bromide staining. After precipitation the sample wastreated with T₄ DNA polymerase and digested with EcoR1 prior toseparation on a 1% agarose gel. The DNA fragments (200 to 400 basepairs) were isolated from the gel, kinased and cloned into pBluescript.Recombinant clones were analyzed by sequencing. One fragment 270 basepairs in length, named S10, was identified. This sequence contained a“TM IV” like domain and represented a potentially new serotoninreceptor. The corresponding full length cDNA was isolated from a ratbrain cDNA library.

[0095] Rat PCR primers (from TM3 to TM7) were used to amplifysingle-stranded cDNA prepared from human heart, brain and retina, asdescribed above. Those human PCR DNA fragments were subcloned inpBluescript and sequenced.

[0096] cDNA Library Construction, screening and Sequencing: Rat brainswere dissected from adult male CD rats (Charles River Laboratories) andtotal RNA was prepared by the guanidine thiocyanate method (Chirgwin, J.W. et al.: 1979). Poly A⁺RNA was purified with a Fast track kit(Invitrogen Corp., San Diego, Calif.). Double stranded (DS) cDNA wassynthesized from 5 μg of poly A⁺RNA according to Gubler and Hoffman(Gubler, U. and B. J. Hoffman, 1983). The resulting DS cDNA was ligatedto BstxI/EcoRI adaptors (Invitrogen Corp.), the excess of adaptors wasremoved by chromatography on Sepharose CL 4B (Pharmacia LKB) and the DNAwas then size selected on a Gen-Pak Fax HPLC column (Zhao, D. et al.,1992) (Waters, Millipore Corp., Milford, Mass.). High molecular weightfractions were ligated in pCDM8 cut by BstxI (Invitrogen Corp.). Theligated DNA was electroporated in E.Coli MC 1061 (Gene Pulser, Biorad).A total of 20×10⁶ independent clones with an insert mean size of 1.9 kbcould be generated. Before amplification, the library was divided intopools of 2.5 to 5×10⁴ independent clones. After 18 hours amplification,the pools were stored at −85° C. in 20% glycerol.

[0097] 100 pools of the cDNA library, representing 3.2×10⁶ primaryclones, were screened using exact PCR primers derived from the S10 PCRclone sequence. 1 μl (4×10⁶ bacteria) of each amplified pool wassubjected directly to 40 cycles of PCR and the resulting productsanalyzed by agarose gel electrophoresis and Southern blotting. Two outof four positive pools were analyzed further and by sib selection andplating out, two individual full length cDNA clones, S10-87 and S10-95,were isolated. DS-DNA was sequenced with a sequanase kit (USBiochemical, Cleveland, Ohio) according to the manufacturer. Nucleotideand peptide sequences analysis were performed with GCG programs.

[0098] Genomic Cloning and Sequencing: A human fibroblast genomiclibrary in λ dash II (≈1.5×10⁶ total recombinants; Stratagene, LaJolla,Calif.) was screened using a 45 nt. oligonucleotide probe derived fromthe rat S10-87 receptor gene, designed in the 3′ end of the carboxylterminal tail (from the anti-sense strand [nucleotide 1220-1264), 5′TCAAAAGCATGATTCCAGGGACTCTGGGTCATTGTGTATGGGCAA 3′ (SEQ ID No. 11) (seeFIG. 1). The oligomer was labeled with [³²P]γATP by using polynucleotidekinase. Hybridization was performed at medium stringency conditions: 45°C. in a solution containing 37.5% formamide, 5×SSC (1×SSC is 0.15Msodium chloride, 0.015M sodium citrate), 1×Denhardt's solution (0.02%polyvinylpyrrolindone, 0.02% Ficoll, 0.02% bovine serum albumin), and200 μg/μl sonicated salmon sperm DNA. The filters were washed at 45° C.in 0.1×SSC containing 0.1% sodium dodecyl sulfate and exposed at −70° C.to Kodak XAR film in the presence of an intensifying screen. Lambdaphage clones hybridizing with the probe were plaque purified and DNA wasprepared for Southern blot analysis (Southern, 1975; Sambrook et al.,1989). A 900 bp Hind2/SstI hybridizing fragment was subcloned into pUC18(Pharmacia, Piscataway, N.J.)). Nucleotide sequence analysis wasaccomplished by the Sanger dideoxy nucleotide chain termination method(Sanger et al., 1977) on denatured double-stranded plasmid templates,using Sequenase (US Biochemical Corp., Cleveland, Ohio).

[0099] PCR amplification of a partial length human 810-87 cDNA clone:

[0100] The 900 bp Hind2/SstI fragment contained sequence encoding thehuman S10-87 carboxy terminal tail, including the stop codon. Thissequence was used to generate a 25 mer (reverse primer) containing thestop codon: 5′ CCTCAATCAGAAGCATGATTCCAGG 3′ (SEQ ID No. 12). As aforward primer we used the 5′ end of the human PCR fragment previouslyidentified (FIG. 6): 5′TTGGTCTATAGGAACAAGATGACCC 3′ (SEQ ID No. 13).These human PCR primers were used to amplify single stranded cDNAprepared from human brain as previously described. The amplified DNA wassubcloned and sequenced as described above.

[0101] Isolation of the full length human S10-95 cDNA clone: 20 pools ofa human hippocampal cDNA library (3 kb average size insert, in pcEXV-3)representing 10⁶ independent clones were screened by PCR with TM4-TM6primers as previously described. Five positive pools were identified.One of those pools was analyzed further and by sib selection a 5 kb cDNAclone, CG-17, was isolated. Double Stranded-DNA was sequenced asdescribed above. Nucleotide and peptide sequence analysis were performedwith the Genetics Computer Group sequence analysis software package.

[0102] DNA transfection: The full coding region of S10-87 (clone CG-5)and S10-95 (clones CG-6 and CG-17) were subcloned in the correctorientation in the mammalian expression vectors pCDNA1-Amp (InvitrogenCorp.), and pcEXV-3 (Miller, J. and R. N. Germain, 1986) (CG-7 and CG-8respectively). For transient expression, Cos-7 cells were transfected bythe DEAE-Dextran method, using 1 μg of DNA /10⁶ cells (Warden, D. and H.V. Thorne, 1968).

[0103] Membrane Preparation: Membranes were prepared from transientlytransfected COS-7 cells which were grown to 100% confluence. The cellswere washed twice with phosphate-buffered saline, scraped from theculture dishes into 5 ml of ice-cold phosphate-buffered saline, andcentrifuged at 200×g for 5 min at 4′. The pellet was resuspended in 2.5ml of ice-cold Tris buffer (20 mM Tris -HCl, pH 7.4 at 23°, 5 mM EDTA),and homogenized by a Wheaton tissue grinder. The lysate was subsequentlycentrifuged at 200×g for 5 min at 4° to pellet large fragments whichwere discarded. The supernatant was collected and centrifuged at40,000×g for 20 min at 4°. The pellet resulting from this centrifugationwas washed once in ice-cold Tris wash buffer and finally resuspended ina final buffer containing 50 mM Tris-HCl and 0.5 mM EDTA, pH 7.4 at 23°.Membrane preparations were kept on ice and utilized within two hr forthe radioligand binding assays. Protein concentrations were determinedby the method of Bradford (1976) using bovine serum albumin as thestandard.

[0104] Radioligand Binding: [²H]5-HT binding was performed using slightmodifications of the 5-HT_(1D) assay conditions reported byHerrick-Davis and Titeler (1989) with the omission of masking ligands.Radioligand binding studies were achieved at 37° C. in a total volume of250 μl of buffer (50 mM Tris, 10 nM MgCl₂, 0.2 mM EDTA, 10 μM pargyline,0.1% ascorbate, pH 7.4 at 37° C.) in 96 well microtiter plates.Saturation studies were conducted using [³H]5-HT at 10 differentconcentrations ranging from 1.0 nM to 100 nM. Displacement studies wereperformed using 10 nM [³H]5-HT. The binding profile of drugs incompetition experiments was established using 7 concentrations ofcompound. Incubation times were 30 min for both saturation anddisplacement studies. Nonspecific binding was defined in the presence of10 μM 5-HT. Binding was initiated by the addition of 50 μl membranehomogenates (10-20 μg). The reaction was terminated by rapid filtrationthrough presoaked (0.5% polyethyleneimine) filters using 48R CellBrandel Harvester (Gaithersburg, Md.). Subsequently, filters were washedfor 5 sec with ice cold buffer (50 mM Tris HCL, pH 7.4 at 4° C.), driedand placed into vials containing 2.5 ml of Readi-Safe (Beckman,Fullerton, Calif.), and radioactivity was measured using a Beckman LS6500C liquid scintillation counter. The efficiency of counting of[³H]5-HT averaged between 45-50%. Binding data were analyzed bycomputer-assisted nonlinear regression analysis (Accufit and Accucomp,Lundon Software, Chagrin Falls, Ohio). IC₅₀ values were converted toK_(f) values using the Cheng-Prusoff equation (1973).

[0105] [³H]GR113808 binding was performed using slight modifications ofthe method of Waeber et al., 1993. Radioligand binding studies wereachieved at 37° C. in a total volume of 250 μl of buffer (50 mM Tris, 10μM, 0.01% ascorbate, pH 7.4 at 37° C.) in 96 well microtiter plates.Saturation studies were conducted using [³H]GR113808 at 10-12 differentconcentrations ranging from 0.005-2.5 nM. Displacement studies wereperformed using 0.2-0.4 nM [³H]GR113808. The binding profile of drugs incompetition experiments was established using 10-12 concentrations ofcompound. Incubation times were 30 min for both saturation anddisplacement studies. Nonspecific binding was defined in the presence of50 μM 5-HT. Binding was initiated and terminated as described for[³H]5-HT binding (see above). Radioactivity was measured and data wereanalyzed as described above for [³H]5-HT.

[0106] Measurement of cAMP Formation: The transiently transfected Cos-7cells were incubated in Dulbecco's modified Eagle's medium, 5 mMtheophylline, 10 mM Hepes (4-[2-Hydroxyethyl]-1-piperazineethanesulfonicacid), 10 μM pargyline, and/or appropriate concentrations of antagonistsfor 20 minutes at 37° C., 5% CO₂. Serotonin or other agonists in thepresence or absence of forskolin (FSK) (10 μM) were then added atappropriate concentrations and incubated for an additional 10 minutes at37° C., 5% CO₂. The media was aspirated and the reaction stopped by theaddition of 100 mM HCl. The plates were stored at 4° C. for 15 minutes,centrifuged for 5 minutes, 500×g to pellet cellular debris, and thesupernatant aliquotted and stored at −20° C. prior to assessment of cAMPformation by radioimmunoassay (cAMP Radioimmunoassay kit, AdvancedMagnetics, Cambridge, Mass.). Radioactivity was quantitated using aPackard COBRA Auto Gamma Counter equipped with data reduction software.Functional data was fitted to a four parameter logistic equation toobtain response parameters (EC₅₀, E_(max), nH; Inplot, GraphPad, SanDiego, Calif.).

[0107] Drugs: [³H]5-HT (specific activity=22.7 Ci/mmole) was obtainedfrom New England Nuclear, Boston, Mass. [³H]GR113808 (specificactivity=82 Ci/mmol) was obtained from Amersham International (ArlingtonHts., Ill.). All other chemicals were obtained from commercial sourcesand were of the highest grade purity available.

[0108] Experimental Results

[0109] A 270 bp DNA fragment (S10) was identified when rat brain cDNAwas used as template in a PCR amplification with two degenerateoligonucleotide primers derived from well conserved regions amongseveral serotonin receptors, in the third and fifth putativetransmembrane domains. The peptide sequence corresponding to this S10PCR clone contained a “transmembrane IV like” domain. Since we used“serotonin receptor specific” PCR primers, this S10 clone represented apotentially new serotonin receptor. The corresponding full length cDNAwas isolated from a rat brain cDNA library. Since five amplifiedcommercial phage cDNA libraries turned out to be negative, we split theplasmid cDNA library into small pools of 2.5 to 5×10⁴ independent clonesbefore amplification to avoid a potential growth bias against the S10cDNA clone. By direct PCR analysis of bacterial pools, subsequent sibselection and standard filter hybridization, two cDNA clones wereidentified, S10-87 (5.5 kb) and S10-95 (4.5 kb). The nucleotide anddeduced amino acid sequences are shown in FIG. 1 (S10-87) and FIG. 2(S10-95). Surprisingly the peptide sequences between those two clonesare only 96.7% identical, diverging in the second half of the carboxyterminus tails, downstream of position 359 (FIG. 3). In addition, theentire 3′ untranslated regions are totally divergent. The longest openreading frame for S10-87 encodes a protein of 387 amino acids and 406amino acids for S10-95. The hydrophobicity plot displayed sevenhydrophobic, putative membrane spanning regions which when compared toother G protein-coupled receptors did not show any significant highhomologies, even to other serotonin receptors (Table 1 and FIG. 4). Itis interesting to note that the highest homology, overall or restrictedto the 7 TM region, is exhibited by the human histamine H₂ receptor,which like the 5-HT₄ receptor, is coupled to stimulation of cAMP.

[0110] Both S10-87 and S10-95 proteins carry 4 potential N-glycosylationsites in positions 7, 180, 316, and 352. They also possess 3 potentialphosphorylation sites for protein kinase C in positions 218, 248, 318and 4 potential phosphorylation sites for casein kinase II in positions9, 97, 218 and 288. A potential palmitoylation site is present in bothclones at the cysteine found in position 329. A large number of Gprotein-coupled receptors carry a cysteine in the same position andO'Dowd et al. have speculated that it plays an important role in thefunctional coupling of the human β₂-adrenergic receptor. In addition,clone S10-95 carries one more potential phosphorylation site for proteinkinase C at position 400. This additional phosphorylation site couldlead to differential functional coupling between the S10-87 and S10-95receptors.

[0111] Since we isolated two different S10 cDNA clones by screening alibrary made from an entire brain, we checked for differentialexpression in seven different parts of the brain by PCR amplificationusing pairs of primers specific for each clone. The results aresummarized in table 2. Clone S10-95 seems to be transcribed everywherein the rat brain except in cerebellum. Clone S10-87 is only expressed instriatum. It remains to be determined if only one or both receptors areexpressed in rat cortex.

[0112] The partial human S10-87 nucleotide (FIG. 11A) and deduced aminoacid sequences (FIG. 11B) are shown. The sequences are highly similar tothe rat S10-87 homolog, 90.8% at the nucleotide level and 93.8% at theamino acid level (FIGS. 12 and 13 respectively).

[0113] The full length human S10-95 nucleotide (FIG. 14A) and deducedamino acid sequences (FIG. 14B) are shown. Compared to the rat S10-95sequence, it shows 90.7% identity at the nucleotide level and 91.8%identity at the amino acid level (FIGS. 15 and 16 respectively). Thehuman S10-95 nucleotide sequence contains one nucleotide insertion inposition 1159. This insertion creates a frame shift and introduces astop codon in the reading frame 16 nucleotides downstream. The proteinmotifs are highly conserved between the rat and human homologs exceptfor a casein kinase II potential phosphorylation site in position 288which is lost in both human receptors. The human homologs both carry apotential cAMP/cGMP phosphorylation site in position 338 in theircarboxy terminal tail which is absent in the rat homologs. A comparisonof the amino acid sequence between the human and the rat S10-95 clonesbeginning from the initiating methionine and ending with the stop codonof the human S10-95 clone, reveals 31 amino acid changes of which 11 arenon conservative, including 2 in TM1, 1 in TM2 and 1 in TM4. Due to thenucleotide insertion and the corresponding frame shift described above,the carboxy terminal tail of the human S10-95 receptor is 16 amino acidshorter than its rat homolog.

[0114] Identical to the rat homologs, both human clones are identical inthe loops and transmembrane regions, differing only in the second halfof their carboxy terminal tail (FIG. 17, nucleic acid sequence; FIG. 18,aa sequence).

[0115] The human PCR cDNA fragments (TM-3 to TM-7) are 100% identicalbetween heart, brain and retina. The nucleotide and deduced amino acidsequences are shown in FIG. 5. The human sequence shows 90.7% homologywith the rat nucleotide sequence (FIG. 6) and 92.3% homology (FIG. 7)with the rat amino acid sequence.

[0116] The genes encoding the rat S10-87 and S10-95 receptors weretransiently expressed in Cos-7 cells for pharmacological evaluation.Initial experiments using 5 nM [³H]5-HT indicated that both S10-87 andS10-95 were serotonergic sites as demonstrated by the degree of specificbinding and density of sites expressed in the transfected cells whencompared against the mock transfected controls. Saturation analysis ofS10-87 (CG-7) was performed using 10 concentrations of [³H]5-HT (1-100nM) and yielded a Bmax of 1,938═399 fmol/mg of protein and a K_(d) for[³H]5-HT of 7.87±0.06 nM. The degree of specific binding atconcentrations of [³H]5-HT close to the K_(d) ranged from 70-84%throughout the experimental series (including saturation and competitionstudies). Although the use of [³H]5-HT as a radioligand to label 5-HT₄receptors in brain tissue is not efficient due to the nonselectivity ofthe ligand, it became clear in the present studies using a homogeneousreceptor population that [³H]5-HT would label this particular receptor.In fact, [³H]5-HT appears to be labelling the high affinity state of the5-HT₄ receptor which is not unusual for the conditions upon which thisreceptor has been studied. Similar results using an agonist radioligandhave been previously reported for the cloned 5-HT₂ receptor (Branchek etal., 1990).

[0117] A pharmacological binding profile of S10-87 and S10-95 (CG-7 andCG-8) was performed and demonstrated that this novel receptor wassimilar to the 5-HT₄ receptor as defined by functional assays in theliterature (Bockaert et al., 1992). This is clearly shown in table 3where the binding affinities of various serotonergic agents aredisplayed for S10. Notably, 5-HT and the tryptamine derivative5-methoxytryptamine possessed high affinity. Furthermore, as previouslyreported for the 5-HT₄ receptor, benzamide derivatives includingcisapride, BRL 24924 and zacopride bound with fairly high affinity toreceptors expressed from the S10 gene. ICS 205930, a tropanyl-indolederivative, which has been reported to be an antagonist at both 5-HT₃and 5-HT₄ receptors (Bockaert et al., 1992), also bound to thesereceptor sites. Compounds such as8-hydroxy-2-(di-n-propylamino)tetralin, ketanserin, sumatriptan and5-carboxyamidotryptamine were of low affinity having K_(f) valuesestimated to be greater than 1 μM. This data would rule out S10 frombelonging to other serotonergic receptor subfamilies such as 5-HT₁ and5-HT₂. Taken together, the complete pharmacological profile alsodifferentiates S10 from the related subtype 5-HT_(4B) (U.S. Ser. No.,971,960, filed, Nov. 3, 1992, copending). Although some of the drugstested also have good affinity for 5-HT₃ receptors, S10 is clearly a5-HT₄ receptor based upon the binding data and the functional datademonstrating a positive-coupling to adenylate cyclase. Finally, acorrelation plot for the binding affinities of 5-HT, cisapride, BRL24924, zacopride, and ICS 205930 against their functional responses inadenylate cyclase assays from cultured mouse collicular neurons (Dumuiset al., 1989) yielded a correlation coefficient of 0.96 (FIG. 8). Thus,the rank order of potency for these key compounds also providesconclusive evidence that S10 encodes a 5-HT₄ receptor.

[0118] To examine the ability of S10 clone to couple to adenylatecyclase, Cos-7 cells transiently expressing S10 were tested for theability to exhibit an increase in basal cAMP release or a decrease inFSK-stimulated cAMP response. 5-HT (1 μM) had no effect on either basalor FSK-stimulated adenylate cyclase activity in untransfected ormock-transfected Cos-7 cells (data not shown), indicating thatendogenous cyclase-coupled serotonin receptors are not present inuntransfected cells. Preliminary studies were carried out by adding onlyone dose of various drugs in triplicate. Addition of 5-HT (1 μM) to thissystem caused stimulation of basal cAMP release (CG-7=0.020±0.002;CG-8=0.023±0.004 pmol/ml/10 min) by about 30 fold for each clone; noinhibition of either the basal or FSK-stimulated cAMP release wasobserved. On the contrary, addition of 10 μM FSK together with 1 μM 5-HTstimulated cAMP accumulation about 10-fold more than either agent alone(data not shown). For various compounds, full dose-response curves weredetermined for both clones and the data are summarized in table 4. 5-HTcaused a concentration-dependent stimulation of basal adenylate cyclaseactivity with mean EC₅₀s of 26±3 and 51±7 nM and E_(max)s of 2,107 and2,598% basal cAMP release for CG-7 and CG-8 respectively (FIGS. 9 and10). Among the tryptamine derivatives tested, 5-MeOT was approximatelyequipotent with 5-HT in both clones, whereas α-Me-5-HT and 5-CT wereabout 10 and 200 times respectively less potent than 5-HT at CG-7. Onthe other hand, the latter two compounds displayed approximately 20 and30 fold lower affinity than 5-HT respectively for CG-8. The2-methoxy-4-amino-5-chloro-substituted benzamides (cisapride, BRL-249245and zacopride) were less potent agonists than 5-HT in stimulating basalcAMP release and displayed different rank order of potency for CG-7 andCG-8. As indicated in table 4 using CG-7, cisapride, BRL-24924 andzacopride exhibited approximately 10, 30 and 100 fold lower potency than5-HT respectively, whereas at CG-8 these compounds had almost equalaffinity. Thus, although not different in binding properties, thesesubtle differences in affinity in functional assays of the two“variants” (CG-7 and CG-8) indicate the potential to develop separatetherapeutic entities for each separate target. All the agonists testedacted as full agonists with the exception of cisapride, BRL-24924 andzacopride, which also displayed antagonist activity and were thereforepartial agonists at both clones, with intrinsic activities rangingbetween 0.85 and 1.4 (Table 4). ICS-205-930 (100 μM) had similar effectat the two clones and was found to be a silent antagonist causingparallel dextral shifts in the concentration effect curve of 5-HTwithout altering the maximum response significantly. The estimated K₈value for ICS-205-930 was not significantly different between the twoclones (CG-7=962±244 nM: CG-8=607±30 nM). Responses to 5-HT were notaffected by spiperone or methiothepin (10 μM) in either clone.

[0119] Saturation analysis of rat 5-HT_(4A) S10-87 (CG-7) and S10-95(CG-8) clones and human 5-H^(T) _(4A) clone CG-17 were performed using10-12 concentrations of [³H]GR113808 (0.005-2.5 nM) and revieled asingle saturable site of high affinity for both clones (CG-7: K_(d)=0.74nM, B_(max)=5.7 pmol/mg membrane protein: CG-8: K_(d)=1.0 nM,B_(max)=3.7 pmol/mg membrane protein; CG-17: K_(d)=0.20 nM, B_(max)=1.8pmol/mg membrane protein). These preliminary data indicate that althoughthe rat clones (CG-7 and CG-8) have similar affinity for the antagonist[³H]GR113808, the human clone (CG-17) displays approximately 5-foldhigher affinity than the rat clones for this ligand. For all threeclones nonspecific binding increased linearly with increasing ligandconcentration. The degree of specific binding at concentrations of[₃H]GR113808 (0.4-0.5 nM) ranged from 80-90%).

[0120] The pharmacological binding profile of S10-87 and S10-95 (CG-7,CG-8, respectively) was investigated in displacement studies using[³H]GR113808 and/or [³H]5-HT. In order to compare CG-17 pharmacologywith that previously obtained for the rat clones, CG-7 and CG-8,displacement studies on the human CG-17 clone were performed using[³H]5-HT as the radioligand.

[0121] A range of 5-HT₄ receptor agonists and antagonists completelyinhibited the specific binding of [³H]GR113808 on both the rat CG-7 andCG-8 clones. Affinity values and Hill slopes derived from the curvesusing computer analysis are presented in Table 5. As previously observedusing [³H]5-HT as the radioligand, the rat CG-7 and CG-8 receptors hadvery similar pharmacology. Of the agonists tested, only those active in5-HT₄ containing preparations (5-HT and 5-MeOT) potently inhibited[³H]GR13808. By contrast, agonists for other 5-HT receptors, for example5-HT_(1A) receptor agonist, 8-OH-DPAT, the 5-HT_(1D) receptor agonist,sumatriptan, the 5-HT₂ receptor antagonist, ketanserin, had no effect on[³H]GR113808 binding at concentrations up to 1 μM. The substitutedbenzamides, cisapride, BRL-24924 and zacopride, partial agonists at5-HT₄ receptor all potently inhibited [³H]GR113808 binding. Specific[³H]GR113808 binding was also inhibited by the 5-HT₄ receptor antagonistICS-205930.

[0122] For both the rat CG-7 and CG-8 clones, Hill slopes for theinhibition of [³H]GR113808 binding by 5-HT₄ receptor agonists but notthe antagonist, ICS-205930, were shallow in the absence of Gpp(NH) withthe exception of 5-CT, and α-Me-5-HT. For agonists that had shallowdisplacement curves, the binding was resolved into high and low affinitycomponents and these are summarized in Table 5. The K_(i) valuesobtained for the high affinity state of the receptor using [³H]GR113808as the radioligand were compatible with the K_(i) values obtainedpreviously using [³H]5-HT as the radioligand which labels the highaffinity state of the receptor (Table 5 and 6). Some differences wereobserved for the K_(i) values of high affinity state of CG-7 compared toCG-8 (Table 3) and their nH values. For example, although there were nodifferences in the K_(i) values of CG-7 and CG-8, the displacement curveobtained for 5-MeOT using CG-8 clone could not be resolved into twosites. Also the K_(i) value obtained for the high affinity state of CG-8using cisapride was approximately 3-fold lower than that obtained forCG-7 previously using [³H]5-HT to directly label the high affinity stateof the receptor. We are currently investigating these differences using[³H]5-HT to directly label the high affinity state.

[0123] In the presence of 100 μM Gpp(NH)p, competition binding curvesfor the agonists displaying shallow curves in the absence of Gpp(NH)pwere shifted to the right and this is exemplified for 5-HT in FIG. TheHill slopes were increased.

[0124] Preliminary results obtained with the human clone (CG-17) using[³H]5-HT as the radioligand in displacement studies are summarized inTable 3. Similar to the rat CG-7 and CG-8 clones, 8-OH-DPAT, sumatriptanand ketanserin were inactive at the CG-17 clone for up to concentrationof 1 μM. The differences observed between the human and the rat CG-8clones were as follows. The biggest difference was observed withα-Me-5-HT which had approximately 100 fold higher affinity for the humanCG-17 clone. Zacopride, 5-MeOT and cisapride had about 7-fold, 5-foldand 4-fold higher affinity, respectively for the human clone CG-17.

[0125] Discussion

[0126] We have identified two cDNA clones encoding thepharmacologically-defined 5-HT₄ receptor. This receptor is expressed atlow levels in rat brain if we consider its frequency in the cDNA library(≦1:10⁶). Surprisingly, two receptors differing in theircarboxy-terminus regions have been isolated. Since the 3′ untranslatednucleotide sequences are also different, these two receptors could beencoded by two different genes or could arise by alternative splicing ofpre-mRNA. These two receptors (S10-87 and S10-95) are differentiallyexpressed in rat brain and the physiological relevance of the S10-87receptor being expressed only in striatum remains to be determined.

[0127] The pharmacology binding profile and the functional couplingobtained from cells expressing S10 clones indicate that these genes bothencode a pharmacologically-defined 5-HT₄ receptor. The cloned rat CG-7and CG-8 genes transiently expressed in Cos-7 cells coupled tostimulation of adenylate cyclase. The magnitude of this response (−20-25fold) was large. With the exception of 5-MeOT, agonist potenciesdetermined from functional assays were less than expected from K_(i)values obtained from binding assays using [³H]5-HT. Could this result bedue to the possibility that the dose of [³H]5-HT used in binding assaysmeasures only the high affinity site of agonists? This is not likely, asit would not account for the data obtained with ICS-205-930 which is asilent antagonist in the present system displaying approximately 6(CG-8) and 10 (CG-7) fold lower affinity in the functional assay ascompared to the binding experiments. It is more likely that differencesin experimental conditions used in binding assays compared with thoseused in the functional assays (such as membrane vs. cells, buffers andextent of equilibrium achieved) accounts for these differences.

[0128] 5-HT responses were resistant to blockade by methiothepin andspiperone (10 μM). As the concentration of these agents exceed theirequilibrium dissociation constants for their respective receptor sitesby 10-100 fold, it seems that 5-HT₁-like (5-HT_(1A), 5HT_(1B), 5BH_(1C),5HT_(1D), 5HT_(1E), 5-HT_(1F)), 5-HT₂ and 5-HT_(4B) receptors can beruled out. In addition, the weak agonistic activity of 5-CT relative to5-HT further supports the notion that 5-HT₁-like receptors are probablynot involved (Bradley, 1986). The results obtained with the indoleagonists reflect those reported at the 5-HT₄ receptor in both the CNSand the periphery (Dumuis et al., 1988; Craig and Clarke, 1990; Eglen etal., 1990; Baxter, Craig and Clarke, 1991). The substituted benzamides,cisapride, BRL-24924 and zacopride acted as partial agonists. Althoughthe benzamides also possess affinity for 5-HT₃ receptors, they lackintrinsic efficacy (Schuurkes et al., 1985; Sanger and King, 1988).Furthermore, the affinity of ICS-205-930 for antagonism of 5-HT responseat S10 is 1-3 orders of magnitude lower than that at 5-HT₃ receptors(Richardson et al., 1985) and therefore indicates a binding sitedifferent from 5-HT₃ receptor.

[0129] As was the case with the rat 5-HT₄ receptor, there are two formsof the human homolog, most likely splice variants differing in thecarboxyterminal end of the receptor. Non conservative amino acidsubstitutions, especially in transmembrane domains 1, 2 and 4 couldprovide the basis for the pharmacological differences observed betweenthe rat and the human 5-HT₄ receptors (see below). A difference in thefunctional response is observed between the two rat clones: although theCG-7 construct (S10-87) gives higher levels of receptor expression inCOS-7 cells (Bmax of 5.7 pmol/mg of protein versus 3.7 pmol/mg forS10-95), in the functional assay, the CG-8 construct (S10-95)reproducibly shows a higher level of cAMP stimulation (2598±154% ofbasal cAMP release versus 2107±52% for CG-7). This finding could beattributed to the different amino acid sequence in the carboxy terminaltail of the rat receptors, specially since the rat S10-95 isoformcarries an additional potential phosphorylation site at position 400,absent in S10-87 (CG-7).

[0130] Since the human S10-95 homolog lacks the last 16 carboxy terminalamino acids which carry the phosphorylation site mentioned above in therat homolog, it will be interesting to check for differences in thelevel of cAMP stimulation upon activation of the rat and human S10-95homologs. In the same way, after we get the full length human S10-87,both human isoforms will be compared in binding and functional assays.

[0131] All the unique pharmacological characteristics described abovedefine the S10 genes as adenylate cyclase stimulatory “5-HT₄” receptorsreported in the literature that are expressed in the ileum (Craig andClarke, 1990), hippocampus (Shenker et al., 1987), esophagus (Baxter etal., 1991), embryo colliculi neurons (Dumius et al., 1988), atrium(Kaumann et al., 1990), adrenal (Lefebvre et al., 1992) and bladder(Corsi et al., 1991), and distinguish these clones from all other clonedsubtypes of 5-HT receptor. Although the binding profile of CG-7 and CG-8were identical (Table 3), some differences in agonist potency(benzamides in particular) were observed between them in the functionalassays. This is not surprising since the amino acid sequences of thesetwo clones are identical, apart from the cytoplasmic carboxy tail, aregion that is important for G protein-coupling, where the CG-8 receptorcarries an extra phosphorylation site. Cisapride, BRL-24924 andzacopride had similar affinities at CG-8 whereas BRL-24924 and zacopridedisplayed approximately 4 and 15 fold lower affinity than cisapride atCG-7 clone for stimulation of adenylate cyclase. It is noteworthy thattissue differences in the potency of benzamides have been reported(Baxter et al., 1991) and whether this reflects a heterogeneity of 5-HT₄receptors remains to be investigated.

[0132] The data obtained with the rat CG-7 and CG-8 clones and the humanCG-17 clone using [³H]GR113808 are very similar to those reported byGrossmann et al. (1993) and Waeber et al. (1993) with this ligand usingguinea pig and human brain tissues. Specific [³H]GR113808 bindingreadily saturated for all three clones (CG-7, CG-8 and CG-17). Scatchardanalysis of specific binding in all three clones revealed theinvolvement of a single site. Curve fitting analysis showed anequilibrium dissociation of approximately 1 nM for both rat clones (CG-7and CG-8) whereas this value was about 5-fold lower for the human CG-17clone (K_(d)=0.2 nM). The K_(d) value obtained for the human CG-17 cloneis in excellent agreement with that reported by Waeber et al. (1993)using human brain (0.23-0.37 nM) and is very similar to that of theguinea pig brain tissue (0.13-0.2 nM: Waeber et al., 1993; Grossmann etal., 1993). The K_(d) value of [³H]GR113808 for rat brain tissue has notbeen reported, however, it is interesting that the affinity obtained forGR113808 from functional receptor assays in the rat oesophagus(Grossmann et al., 1993) is about 0.3 nM which indicates that thisantagonist has similar affinities for the human and the rat tissue used.The discrepancies between our data and those reported may be due tomethodology, or different subtype (brain vs. oesophagus), however, thisremains to be investigated.

[0133] The rank order of potency of compounds competing for specific[³]GR113808 are very similar for both CG-7 and CG-8 and iscisapride>5-HT>BRL-24924>5-MeOT=ICS205930>zacopride>α-Me-5-HT>5-CT. Thisorder of potency is different from that observed with guinea pig caudate(Grossmann et al., 1993;cisapride>5-HT>ICS205930>BRL-24924>zacopride>5-MeOT>α-Me-5-HT>5-CT) andhuman caudate (Waeber et al., 1993;cisapride>ICS205930>BRL-24924>5-HT>5-MeOT. Whether these differences aredue to species or different population of high affinity state of thereceptor in the various preparation, remains to be investigated.Interestingly, the displacement curves for most of the agonistscompeting for specific [³H]GR113808 were shallow and could be resolvedto high and low affinity states. Gpp(NH)p shifted these curves to theright and in the case of 5-HT the Hill coefficient was increased tounity; however for some agonists the shift was not complete. Grossmannet al.(1993) using guinea pig caudate also observed shallow competitioncurves for some agonist that could be partially shifted by the additionof GTP (Grossmann et al., 1993). However, Waeber did not observe shallowbinding curves with the human caudate tissue. These observationsindicate that the G protein content of these prepartations may bedifferent which may reflect differences in the coupling of the receptorwith a G protein.

[0134] Using [³H]5-HT as the radioligand, the affinity values for thehuman CG-17 clone are in general comparable with that obtained by uspreviously tor the rat CG-7 and CG-8 clones with few exceptions. Themost striking differences appears to be for α-Me-5-HT which displaysapproximately 100 fold higher affinity for the human CG-17. However, ithas to pointed out that the data are compared with 2 differentradioligands and this difference has to be further investigated usingthe same radioligand for all both the rat and human clones in parallel.5-MeOT was approximately 3 fold less potent whereas zacopride was about7 fold more potent at the human CG-17 as compared to the rat clones.

[0135] The cloning and expression of genes encoding 5-HT₄ receptorsallows, for the first time, the ability to develop subtype selectivedrugs using radioligand binding assays. It will further providedefinitive answers to whether there are significant species differencesin the pharmacology of 5-HT₄ receptors. In addition, the intrinsicactivity of drugs can be determined from measures of adenylate cyclaseactivation in these transfected cells. So far, native tissuepreparations have shown great disparity in agonist activity. 5-HT₄receptors have been implicated in a wide variety of functions. Existingdrugs such as metaclopramide and cisapride appear to exert a large partof their action through 5-HT₄ receptors (Taniyama et al., 1991;Meulemans and Schurkes, 1992; Flynn et al., 1992). Experience with theseagents indicates a clear therapeutic role for 5-HT₄ receptors in thegastrointestinal system for conditions including irritable boweldisease, postoperative ileus, diabetic gastroparesis, emesis, achalasia,hiatal hernia, and esophageal spasm. In addition, 5-HT₄ receptors havebeen described functionally in the heart (Kaumann, 1992), adrenal(Lefebvre et al., 1992), and bladder (Corsi et al., 1991) indicatingpossible roles in cardiac rate and force of contraction, endocrinecontrol of cortisol secretion, and urinary incontinence or spasticity.5-HT₄ receptors have also been described in the brain, particularly inareas such as the hippocampus, in which we have localized the geneencoding 5-HT₄ receptors (S10-87), indicating a potential role incognitive enhancement (Bockaert et al., 1992). As more specificpharmacological tools are developed, additional therapeutic indicationswill certainly be uncovered. TABLE I % TM homology of the S10 receptorwith other 7 TM receptors SEROTONIN ADRENERGIC DOPAMINE PEPTIDE5-HT_(1A) Hu 44 Alpha-1A Hu 45 D₁ Hu 43 Subst K 25 Hu 5-HT_(1Dα) Hu 40Alpha-1B Hu 43 D₂ Hu 42 TSH 27 5-HT_(1Dβ) Hu 41 Alpha-1C Hu 40 D₃ Rt 465-HT_(1E) Hu 41 Alpha-2A Hu 42 D₄ Hu 45 5-HT_(1F) Hu 41 Alpha-2B Hu 40D₅ Hu 45 5-HT₂ Hu 42 Alpha-2C Hu 40 5-HT_(1C) Hu 44 Beta-1 Hu 46HISTAMINE MUSCARINIC 5-HT Dro S 43 Beta-2 Hu 44 5-HT Dro I_(A) 40 Beta-3Hu 42 H₁ Bov 36 m1 35 5-HT Dro I_(B) 41 H₂ Hu 46 5-HT_(4B) Hu 44ADENOSINE A1 Rat 32 A2 Hu 31

[0136] TABLE 2 Table 2: PCR localization of the S10 mRNA in 7 differentpart of the rat brain The TM3-5 primers do not differentiate betweenclones S10-87 and S10-95. The S10-87 primers were designed from thenucleotide sequences coding for the TM 6 domain common to both receptorsand for the carboxy terminus end specific to S10-87. In the same way,the S10-95 primers are specific for S10-95. Corte Cerebell BrainHippocamp Olfactory Striatu Thalam Primers x um Stem us Bulb m usTH3-5 + − + + + + + S10-97 ND − − − − + − S10-95 ND − + + + + +

[0137] TABLE 3 Binding affinities of key ligands for the identificationof S10 (CG-7 and CG-8) as a 5-HT₄ receptor. Affinity constants (Ki; nM)of drugs competing for S10 labeled with 10 nK [3H⁴]5-HT were determinedto pharmacologically define the encoded receptor as 5-HT₄. Ki (nM)values were calculated using the Cheng-Prusoff equation or estimated tobe >1000 nM based upon one point displacements using a drugconcentration of 1 uM. Affinity constants are expressed as the mean ±SEM (n ≦ 2). Ki values estimated to be >1000 were determined accordingto one point displacements studies at a concentration of 1 uM. (n = 2except BRL 24924 tested at CG-8: n = 1) CHARACTERIZATION OF CLONE S-10Saturation Analysis: Kd = 7.87 ± 0.06 nM Bmax = 1,938 fmol/mg protPharmacological profile: DRUG CG-7 CG-8 5-HT 8.6 ± 0.6 6.4 ± 0.5Cisapride 10.9 ± 0.3  ND 5-MeOT 27.5 ± 5   ND BRL 24924 27.7 ± 5   21.1*ICS 205930 115 ± 12  138 ± 26  Zacopride 130 ± 10  136 ± 5 BOHDPAT >1000 ND Ketanserin >1000 ND Sumatriptan >1000 ND 5-CT >1000 ND

[0138] TABLE 4 Pharmacological profile for the cAMP response using thehuman 5-Ht_(4A) (CG-7 and CG-8) receptor transiently expressed in Cos-7cells, comparison with the binding data obtained with CG-7 clone using[³H]5-HT. cAMP measurements on intact cells were as described underMethods and Materials EC₅₀ values (concentration producing thehalf-maximal effect) were derived from the analysis of fulldose-response curves. Maximum response produced by each drug wasnormalized to the 5-HT maximum response which is indicated as having anintrinsic activity of 1.0. Data are means ± S.E.M. of three separateexperiments. The apparent dissociation constant of antagonist (K_(s))(ICS 205930) was calculated according to the formula: K_(s) =[B]/(A′/A)-1], where [B] is the concentration of antagonist, A′ and Athe EC₅₀ values of agonist measured respectively in the presence and inthe absence of antagonist (Furchgott, 1972). EC₅₀ or K_(s) (nM) I. A. *K₁ (nM) K₁ (nM) DRUG CG-7 CG-8 CG-7 CG-8 CG-7 CG-8 5-MeOT  21 ± 6   31 ±13 1.0 1.0 27 ± 5 ND 5-HT  26 ± 3  51 ± 7 1.0 1.0  8.6 ± 0.6  6.4 ± 0.5Cisapride  191 ± 26   413 ± 199 1.2 1.4   11 ± 0.3 ND α-Me-5-HT  250 ±91 1,038 ± 31  0.90 1.0 ND ND BRL-24924   736 ± 129  250 ± 25 1.1 0.9 28± 5 21 Zacopride 2,740 ± 274  239 ± 33 1.1 1.0 130 ± 10 136 ± 5  5-CT5,570 ± 808 1,411 ± 211 0.85 1.2 >1,000 ND ICS-205930   962 ± 244  607 ±30 0 0 115 ± 12 138 ± 26

[0139] TABLE 5 The affinities of various compounds that compete for0.2-0.4 nM [³H] GR113808 binding in membranes of COS-7 cells transientlytransfected with rat clones CG-7 and CG-8. COM- POUNDS CG-7 CG-8 5-HT237 K_(H) = 2.6, BH = 22% 116 KH = 2.5, BH = 24% K_(L) = 357 KL = 197 nH= 0.62 nH = 0.67 5-CT >10,000 >10,000 5-Me-OT 438 KH = 14, BH = 17% 518KH = , BH = KL = 658 KL = nH = 0.57 nH = 0.66 BRL- 189 KH = 37, BH = 34%188 KH = 23, BH = 32% 24924 KL = 371 KL = 373 nH = 0.85 nH = 0.81 ZACO-729 KH = 424, BH = 68% 820 KH = , BH = PRIDE KL = 2,757 KL = nH = 0.80nH = 0.85 d-LSD >10,000 >10,000 CISA- ND  84 KH = 2.7, BH = 11% PRIDE KL= 105 nH = 0.83 ICS- ND 529 205930 α-Me- 2,255 1,855 5-HT

[0140] TABLE 6 Binding affinities of key ligands for the identificationof the human CG-17 clone as a 5-HT_(4A) receptor. COMPOUND K_(i) (nM)5-HT 4.2 5-MeOT 71 5-CT >10,000 Cisapride 12 α-Me-5-HT 1.6 BRL-24924 21Zacopride 17 Sumatriptan >1,000 8-OH-DPAT >1,000 Ketanserin >1,000

[0141] Affinity constants (Ki, nM) of drugs competing for CG-17 clonedlabeled with 10 nM [³H]5-HT were calculated using the Cheng-Prusoffequation or estimated to be greater than 1,000 nM based upon one pointdisplacement using a drug concentration of 1 μM. Values are from asingle experiment.

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1 19 1 1642 DNA Rattus norvegicus 1 agccttgccg agcctggctt ggttggaaggaggaggatgc tctgcgtgcc cagggtcctg 60 tgggcactga catccaacgt actcatgcccatttcctgta atggacagac ttgatgctaa 120 tgtgagttcc aacgagggtt tcgggtctgtggagaaggtc gtactgctca cgttcttcgc 180 aatggttatc ctgatggcca tcctgggcaacctgctggtg atggttgctg tgtgcaggga 240 caggcagctc aggaaaataa aaaccaattatttcattgtg tctcttgcct ttgctgatct 300 gctggtttcg gtgctggtga atgccttcggtgccattgag ttggtccaag acatctggtt 360 ttatggggag atgttttgcc tggtccggacctctctggat gtcctactca ccacagcatc 420 aatttttcac ctctgctgca tttccctggataggtattat gccatctgct gtcaaccttt 480 ggtttataga aacaagatga cccctctacgcatcgcatta atgctgggag gctgctgggt 540 cattcccatg tttatatctt ttctccccataatgcaaggc tggaacaaca tcggcatagt 600 tgatgtgata gagaaaagga aattcaaccacaactctaac tctacattct gtgtcttcat 660 ggtcaacaag ccctatgcca tcacctgctctgtggtggcc ttctacatcc cgtttctcct 720 catggtgctg gcctattacc gtatctatgtcactgctaag gagcatgccc agcagatcca 780 gatgttacaa cgggcaggag ccacctctgaaagcaggccc cagacagctg accagcacag 840 cacacatcgc atgcggacag agaccaaagcagccaagact ttatgtgtca tcatgggctg 900 cttctgtttc tgctgggccc ccttctttgtcaccaatatt gtggaccctt tcatagacta 960 cactgtgccc gagaaggtgt ggactgctttcctctggctt ggctatatca attcagggtt 1020 gaaccctttt ctctatgcct tcttgaataagtctttcaga cgtgccttcc ttatcatcct 1080 ctgctgtgat gatgagcgct acaaaagaccccccattctg ggccagactg tcccctgttc 1140 aaccacaacc attaatggat ccactcatgtgctaaggtat acagttttgc atagtggtca 1200 acaccaggaa ctggagaagt tgcccatacacaatgaccca gagtccctgg aatcatgctt 1260 ttgattgaag acgtggcttg cctttagcggttcatcccat ctgtgtctgc atgaacaggt 1320 tactatggaa tcactcctga ctctgggcatcaccagtgaa gcatgagcat ggtgaggcag 1380 ggtccggtga aggtgcacag aggacagcattgagtgggac ctgaacccag cacattaagg 1440 atttcagaac cgtgtgggga tttgagatgtcatcagaccc agtgtcttac ccagagccca 1500 actggcacct cccattccac gctgacatgtggtcagtctt tgctcacacc tctccagggg 1560 caggagctga ctacctccta atgtggtggggagctcttaa ttgtgtggaa gttcagtcat 1620 tcattggtgg acagtctcgc tg 1642 2387 PRT Rattus norvegicus 2 Met Asp Arg Leu Asp Ala Asn Val Ser Ser AsnGlu Gly Phe Gly Ser 1 5 10 15 Val Glu Lys Val Val Leu Leu Thr Phe PheAla Met Val Ile Leu Met 20 25 30 Ala Ile Leu Gly Asn Leu Leu Val Met ValAla Val Cys Arg Asp Arg 35 40 45 Gln Leu Arg Lys Ile Lys Thr Asn Tyr PheIle Val Ser Leu Ala Phe 50 55 60 Ala Asp Leu Leu Val Ser Val Leu Val AsnAla Phe Gly Ala Ile Glu 65 70 75 80 Leu Val Gln Asp Ile Trp Phe Tyr GlyGlu Met Phe Cys Leu Val Arg 85 90 95 Thr Ser Leu Asp Val Leu Leu Thr ThrAla Ser Ile Phe His Leu Cys 100 105 110 Cys Ile Ser Leu Asp Arg Tyr TyrAla Ile Cys Cys Gln Pro Leu Val 115 120 125 Tyr Arg Asn Lys Met Thr ProLeu Arg Ile Ala Leu Met Leu Gly Gly 130 135 140 Cys Trp Val Ile Pro MetPhe Ile Ser Phe Leu Pro Ile Met Gln Gly 145 150 155 160 Trp Asn Asn IleGly Ile Val Asp Val Ile Glu Lys Arg Lys Phe Asn 165 170 175 His Asn SerAsn Ser Thr Phe Cys Val Phe Met Val Asn Lys Pro Tyr 180 185 190 Ala IleThr Cys Ser Val Val Ala Phe Tyr Ile Pro Phe Leu Leu Met 195 200 205 ValLeu Ala Tyr Tyr Arg Ile Tyr Val Thr Ala Lys Glu His Ala Gln 210 215 220Gln Ile Gln Met Leu Gln Arg Ala Gly Ala Thr Ser Glu Ser Arg Pro 225 230235 240 Gln Thr Ala Asp Gln His Ser Thr His Arg Met Arg Thr Glu Thr Lys245 250 255 Ala Ala Lys Thr Leu Cys Val Ile Met Gly Cys Phe Cys Phe CysTrp 260 265 270 Ala Pro Phe Phe Val Thr Asn Ile Val Asp Pro Phe Ile AspTyr Thr 275 280 285 Val Pro Glu Lys Val Trp Thr Ala Phe Leu Trp Leu GlyTyr Ile Asn 290 295 300 Ser Gly Leu Asn Pro Phe Leu Tyr Ala Phe Leu AsnLys Ser Phe Arg 305 310 315 320 Arg Ala Phe Leu Ile Ile Leu Cys Cys AspAsp Glu Arg Tyr Lys Arg 325 330 335 Pro Pro Ile Leu Gly Gln Thr Val ProCys Ser Thr Thr Thr Ile Asn 340 345 350 Gly Ser Thr His Val Leu Arg TyrThr Val Leu His Ser Gly Gln His 355 360 365 Gln Glu Leu Glu Lys Leu ProIle His Asn Asp Pro Glu Ser Leu Glu 370 375 380 Ser Cys Phe 385 3 1622DNA Rattus norvegicus 3 agggtcctgt gggcactgac atccaacgta ctcatgcccatttcctgtaa tggacagact 60 tgatgctaat gtgagttcca acgagggttt cgggtctgtggagaaggtcg tactgctcac 120 gttcttcgca atggttatcc tgatggccat cctgggcaacctgctggtga tggttgctgt 180 gtgcagggac aggcagctca ggaaaataaa aaccaattatttcattgtgt ctcttgcctt 240 tgctgatctg ctggtttcgg tgctggtgaa tgccttcggtgccattgagt tggtccaaga 300 catctggttt tatggggaga tgttttgcct ggtccggacctctctggatg tcctactcac 360 cacagcatca atttttcacc tctgctgcct ttccctggataggtattatg ccatctgctg 420 tcaacctttg gtttatagaa acaagatgac ccctctacgcatcgcattaa tgctgggagg 480 ctgctgggtc attcccatgt ttatatcttt tctccccataatgcaaggct ggaacaacat 540 cggcatagtt gatgtgatag agaaaaggaa attcaaccacaactctaact ctacattctg 600 tgtcttcatg gtcaacaagc cctatgccat cacctgctctgtggtggcct tctacatccc 660 gtttctcctc atggtgctgg cctattaccg tatctatgtcactgctaagg agcatgccca 720 gcagatccag atgttacaac gggcaggagc cacctctgaaagcaggcccc agacagctga 780 ccagcacagc acacatcgca tgcggacaga gaccaaagcagccaagactt tatgtgtcat 840 catgggctgc ttctgtttct gctgggcccc cttctttgtcaccaatattg tggacccttt 900 catagactac actgtgcccg agaaggtgtg gactgctttcctctggcttg gctatatcaa 960 ttcagggttg aacccttttc tctatgcctt cttgaataagtctttcagac gtgccttcct 1020 tatcatcctc tgctgtgatg atgagcgcta caaaagaccccccattctgg gccagactgt 1080 cccctgttca accacaacca ttaatggatc cactcatgtgctaagggata cagtggaatg 1140 tggtggccaa tgggagagtc ggtgtcacct cacagcaacttctcctttgg tggctgctca 1200 gccagtgata cgtaggcccc aggacaatga cctagaagacagctgtagct tgaaaagaag 1260 ccagtcctaa gctgctactt cggtgtatgt ggctgcccctggcactttgt tctccaaggc 1320 tttccaagag catgaggcaa tccaccctgg acttcccgccacgattctag caggcggtat 1380 tagaggaagt caggggagag aagggcttcc tccttagctttctgtttctc aacattttct 1440 cttcctggag tctccactct tgcttggtgg tctctgaagtccacgaccca gtcccctttt 1500 gctgtctcca gtctgtcttg taaatgttta ccgtgttcgattttcagttt ccaaacatgc 1560 cttctttgaa gtgtcatctt acgatactgt caaaacatgtgcctgtcttg atcacacttc 1620 tt 1622 4 406 PRT Rattus norvegicus 4 Met AspArg Leu Asp Ala Asn Val Ser Ser Asn Glu Gly Phe Gly Ser 1 5 10 15 ValGlu Lys Val Val Leu Leu Thr Phe Phe Ala Met Val Ile Leu Met 20 25 30 AlaIle Leu Gly Asn Leu Leu Val Met Val Ala Val Cys Arg Asp Arg 35 40 45 GlnLeu Arg Lys Ile Lys Thr Asn Tyr Phe Ile Val Ser Leu Ala Phe 50 55 60 AlaAsp Leu Leu Val Ser Val Leu Val Asn Ala Phe Gly Ala Ile Glu 65 70 75 80Leu Val Gln Asp Ile Trp Phe Tyr Gly Glu Met Phe Cys Leu Val Arg 85 90 95Thr Ser Leu Asp Val Leu Leu Thr Thr Ala Ser Ile Phe His Leu Cys 100 105110 Cys Ile Ser Leu Asp Arg Tyr Tyr Ala Ile Cys Cys Gln Pro Leu Val 115120 125 Tyr Arg Asn Lys Met Thr Pro Leu Arg Ile Ala Leu Met Leu Gly Gly130 135 140 Cys Trp Val Ile Pro Met Phe Ile Ser Phe Leu Pro Ile Met GlnGly 145 150 155 160 Trp Asn Asn Ile Gly Ile Val Asp Val Ile Glu Lys ArgLys Phe Asn 165 170 175 His Asn Ser Asn Ser Thr Phe Cys Val Phe Met ValAsn Lys Pro Tyr 180 185 190 Ala Ile Thr Cys Ser Val Val Ala Phe Tyr IlePro Phe Leu Leu Met 195 200 205 Val Leu Ala Tyr Tyr Arg Ile Tyr Val ThrAla Lys Glu His Ala Gln 210 215 220 Gln Ile Gln Met Leu Gln Arg Ala GlyAla Thr Ser Glu Ser Arg Pro 225 230 235 240 Gln Thr Ala Asp Gln His SerThr His Arg Met Arg Thr Glu Thr Lys 245 250 255 Ala Ala Lys Thr Leu CysVal Ile Met Gly Cys Phe Cys Phe Cys Trp 260 265 270 Ala Pro Phe Phe ValThr Asn Ile Val Asp Pro Phe Ile Asp Tyr Thr 275 280 285 Val Pro Glu LysVal Trp Thr Ala Phe Leu Trp Leu Gly Tyr Ile Asn 290 295 300 Ser Gly LeuAsn Pro Phe Leu Tyr Ala Phe Leu Asn Lys Ser Phe Arg 305 310 315 320 ArgAla Phe Leu Ile Ile Leu Cys Cys Asp Asp Glu Arg Tyr Lys Arg 325 330 335Pro Pro Ile Leu Gly Gln Thr Val Pro Cys Ser Thr Thr Thr Ile Asn 340 345350 Gly Ser Thr His Val Leu Arg Asp Thr Val Glu Cys Gly Gly Gln Trp 355360 365 Glu Ser Arg Cys His Leu Thr Ala Thr Ser Pro Leu Val Ala Ala Gln370 375 380 Pro Val Ile Arg Arg Pro Gln Asp Asn Asp Leu Glu Asp Ser CysSer 385 390 395 400 Leu Lys Arg Ser Gln Ser 405 5 536 DNA Homo sapiens 5ttggtctata ggaacaagat gacccctctg cgcatcgcat taatgctggg aggctgctgg 60gtcatcccca cgtttatttc ttttctccct ataatgcaag gctggaataa cattggcata 120attgatttga tagaaaagag gaagttcaac cagaactcta actctacgta ctgtgtcttc 180atggtcaaca agccctacgc catcacctgc tctgtggtgg ccttctacat cccatttctc 240ctcatggtgc tggcctatta ccgcatctat gtcacagcta aggagcatgc ccatcagatc 300cagatgttac aacgggcagg agcctcctcc gagagcaggc ctcagtcggc agaccagcat 360agcactcatc cgatgaggac agagaccaaa gcagccaaga ccctgtgcat catcatgggt 420tgcttctgcc tctgctgggc accattcttt gtcaccaata ttgtggatcc tttcatagac 480tacactgtcc ctgggcaggt gtggactgct ttcctctggc tcggctatat caattc 536 6 178PRT Homo sapiens 6 Leu Val Tyr Arg Asn Lys Met Thr Pro Leu Arg Ile AlaLeu Met Leu 1 5 10 15 Gly Gly Cys Trp Val Ile Pro Thr Phe Ile Ser PheLeu Pro Ile Met 20 25 30 Gln Gly Trp Asn Asn Ile Gly Ile Ile Asp Leu IleGlu Lys Arg Lys 35 40 45 Phe Asn Gln Asn Ser Asn Ser Thr Tyr Cys Val PheMet Val Asn Lys 50 55 60 Pro Tyr Ala Ile Thr Cys Ser Val Val Ala Phe TyrIle Pro Phe Leu 65 70 75 80 Leu Met Val Leu Ala Tyr Tyr Arg Ile Tyr ValThr Ala Lys Glu His 85 90 95 Ala His Gln Ile Gln Met Leu Gln Arg Ala GlyAla Ser Ser Glu Ser 100 105 110 Arg Pro Gln Ser Ala Asp Gln His Ser ThrHis Pro Met Arg Thr Glu 115 120 125 Thr Lys Ala Ala Lys Thr Leu Cys IleIle Met Gly Cys Phe Cys Leu 130 135 140 Cys Trp Ala Pro Phe Phe Val ThrAsn Ile Val Asp Pro Phe Ile Asp 145 150 155 160 Tyr Thr Val Pro Gly GlnVal Trp Thr Ala Phe Leu Trp Leu Gly Tyr 165 170 175 Ile Asn 7 1316 DNAHomo sapiens 7 cctgtaatgg acaaacttga tgctaatgtg agttctgagg agggtttcgggtcagtggag 60 aaggtggtgc tgctcacgtt tctctcgacg gttatcctga tggccatcttggggaacctg 120 ctggtgatgg tggctgtgtg ctgggacagg cagctcagga aaataaaaacaaattatttc 180 attgtatctc ttgcttttgc ggatctgctg gtttcggtgc tggtgatgccctttggtgcc 240 attgagctgg ttcaagacat ctggatttat ggggaggtgt tttgtcttgttcggacatct 300 ctggacgtcc tgctcacaac ggcatcgatt tttcacctgt gctgcatttctctggatagg 360 tattacgcca tctgctgcca gcctttggtc tataggaaca agatgacccctctgcgcatc 420 gcattaatgc tgggaggctg ctgggtcatc cccacgttta tttcttttctccctataatg 480 caaggctgga ataacattgg cataattgat ttgatagaaa agaggaagttcaaccagaac 540 tctaactcta cgtactgtgt cttcatggtc aacaagccct acgccatcacctgctctgtg 600 gtggccttct acatcccatt tctcctcatg gtgctggcct attaccgcatctatgtcaca 660 gctaaggagc atgcccatca gatccagatg ttacaacggg caggagcctcctccgagagc 720 aggcctcagt cggcagacca gcatagcact catcgcatga ggacagagaccaaagcagcc 780 aagaccctgt gcatcatcat gggttgcttc tgcctctgct gggcaccattctttgtcacc 840 aatattgtgg atcctttcat agactacact gtccctgggc aggtgtggactgctttcctc 900 tggctcggct atatcaattc cgggttgaac ccttttctct acgccttcttgaataagtct 960 tttagacgtg ccttcctcat catcctctgc tgtgatgatg agcgctaccgaagaccttcc 1020 attctgggcc agactgtccc ttgttcaacc acaaccatta atggatccacacatgtacta 1080 agggatgcag tggagtgtgg tggccagtgg gagagtcagt gtcacccgccagcaacttct 1140 cctttggtgg ctgctcagcc cagtgacact taggcccctg ggacaatgacccagaagaca 1200 gccatgcctc cgaaagaggg ccaggtccta agctgctgct tgtgcgcgactgcacccggc 1260 attctcttca cctgaggctt tccgtccgcc agtgcaggaa cccggtgctcgctggg 1316 8 388 PRT Homo sapiens 8 Met Asp Lys Leu Asp Ala Asn Val SerSer Glu Glu Gly Phe Gly Ser 1 5 10 15 Val Glu Lys Val Val Leu Leu ThrPhe Leu Ser Thr Val Ile Leu Met 20 25 30 Ala Ile Leu Gly Asn Leu Leu ValMet Val Ala Val Cys Trp Asp Arg 35 40 45 Gln Leu Arg Lys Ile Lys Thr AsnTyr Phe Ile Val Ser Leu Ala Phe 50 55 60 Ala Asp Leu Leu Val Ser Val LeuVal Met Pro Phe Gly Ala Ile Glu 65 70 75 80 Leu Val Gln Asp Ile Trp IleTyr Gly Glu Val Phe Cys Leu Val Arg 85 90 95 Thr Ser Leu Asp Val Leu LeuThr Thr Ala Ser Ile Phe His Leu Cys 100 105 110 Cys Ile Ser Leu Asp ArgTyr Tyr Ala Ile Cys Cys Gln Pro Leu Val 115 120 125 Tyr Arg Asn Lys MetThr Pro Leu Arg Ile Ala Leu Met Leu Gly Gly 130 135 140 Cys Trp Val IlePro Thr Phe Ile Ser Phe Leu Pro Ile Met Gln Gly 145 150 155 160 Trp AsnAsn Ile Gly Ile Ile Asp Leu Ile Glu Lys Arg Lys Phe Asn 165 170 175 GlnAsn Ser Asn Ser Thr Tyr Cys Val Phe Met Val Asn Lys Pro Tyr 180 185 190Ala Ile Thr Cys Ser Val Val Ala Phe Tyr Ile Pro Phe Leu Leu Met 195 200205 Val Leu Ala Tyr Tyr Arg Ile Tyr Val Thr Ala Lys Glu His Ala His 210215 220 Gln Ile Gln Met Leu Gln Arg Ala Gly Ala Ser Ser Glu Ser Arg Pro225 230 235 240 Gln Ser Ala Asp Gln His Ser Thr His Arg Met Arg Thr GluThr Lys 245 250 255 Ala Ala Lys Thr Leu Cys Ile Ile Met Gly Cys Phe CysLeu Cys Trp 260 265 270 Ala Pro Phe Phe Val Thr Asn Ile Val Asp Pro PheIle Asp Tyr Thr 275 280 285 Val Pro Gly Gln Val Trp Thr Ala Phe Leu TrpLeu Gly Tyr Ile Asn 290 295 300 Ser Gly Leu Asn Pro Phe Leu Tyr Ala PheLeu Asn Lys Ser Phe Arg 305 310 315 320 Arg Ala Phe Leu Ile Ile Leu CysCys Asp Asp Glu Arg Tyr Arg Arg 325 330 335 Pro Ser Ile Leu Gly Gln ThrVal Pro Cys Ser Thr Thr Thr Ile Asn 340 345 350 Gly Ser Thr His Val LeuArg Asp Ala Val Glu Cys Gly Gly Gln Trp 355 360 365 Glu Ser Gln Cys HisPro Pro Ala Thr Ser Pro Leu Val Ala Ala Gln 370 375 380 Pro Ser Asp Thr385 9 31 DNA Artificial Sequence Description of Artificial Sequenceprimer 9 tggaattctg ygynathkcn ctggaymgst a 31 10 27 DNA ArtificialSequence Description of Artificial Sequence primer 10 catnavnrnnarnggdatrw araangc 27 11 45 DNA Artificial Sequence Description ofArtificial Sequence probe 11 tcaaaagcat gattccaggg actctgggtc attgtgtatgggcaa 45 12 25 DNA Artificial Sequence Description of ArtificialSequence primer 12 cctcaatcag aagcatgatt ccagg 25 13 25 DNA ArtificialSequence Description of Artificial Sequence primer 13 ttggtctataggaacaagat gaccc 25 14 792 DNA Homo sapiens 14 ttggtctata ggaacaagatgacccctctg cgcatcgcat taatgctggg aggctgctgg 60 gtcatcccca cgtttatttcttttctccct ataatgcaag gctggaataa cattggcata 120 attgatttga tagaaaagaggaagttcaac cagaactcta actctacgta ctgtgtcttc 180 atggtcaaca agccctacgccatcacctgc tctgtggtgg ccttctacat cccatttctc 240 ctcatggtgc tggcctattaccgcatctat gtcacagcta aggagcatgc ccatcagatc 300 agatgttaca acgggcaggagcctcctccg agagcaggcc tcagtcggca gaccagcata 360 gcactcatcg catgaggacagagaccaaag cagccaagac cctgtgcatc atcatgggtt 420 gcttctgcct ctgctgggcaccattctttg tcaccaatat tgtggatcct ttcatagact 480 acactgtccc tgggcaggtgtggactgctt tcctctggct cggctatatc aattccgggt 540 tgaacccttt tctctacgccttcttgaata agtcttttag acgtgccttc ctcatcatcc 600 tctgctgtga tgatgagcgctaccgaagac cttccattct gggccagact gtcccttgtt 660 caaccacaac cattaatggatccacacatg tactaaggta caccgttctg cacaggggac 720 atcatcagga actcgagaaactgcccatac acaatgaccc agaatccctg gaatcatgct 780 tctgattgag gc 792 15 261PRT Homo sapiens 15 Leu Val Tyr Arg Asn Lys Met Thr Pro Leu Arg Ile AlaLeu Met Leu 1 5 10 15 Gly Gly Cys Trp Val Ile Pro Thr Phe Ile Ser PheLeu Pro Ile Met 20 25 30 Gln Gly Trp Asn Asn Ile Gly Ile Ile Asp Leu IleGlu Lys Arg Lys 35 40 45 Phe Asn Gln Asn Ser Asn Ser Thr Tyr Cys Val PheMet Val Asn Lys 50 55 60 Pro Tyr Ala Ile Thr Cys Ser Val Val Ala Phe TyrIle Pro Phe Leu 65 70 75 80 Leu Met Val Leu Ala Tyr Tyr Arg Ile Tyr ValThr Ala Lys Glu His 85 90 95 Ala His Gln Ile Gln Met Leu Gln Arg Ala GlyAla Ser Ser Glu Ser 100 105 110 Arg Pro Gln Ser Ala Asp Gln His Ser ThrHis Arg Met Arg Thr Glu 115 120 125 Thr Lys Ala Ala Lys Thr Leu Cys IleIle Met Gly Cys Phe Cys Leu 130 135 140 Cys Trp Ala Pro Phe Phe Val ThrAsn Ile Val Asp Pro Phe Ile Asp 145 150 155 160 Tyr Thr Val Pro Gly GlnVal Trp Thr Ala Phe Leu Trp Leu Gly Tyr 165 170 175 Ile Asn Ser Gly LeuAsn Pro Phe Leu Tyr Ala Phe Leu Asn Lys Ser 180 185 190 Phe Arg Arg AlaPhe Leu Ile Ile Leu Cys Cys Asp Asp Glu Arg Tyr 195 200 205 Arg Arg ProSer Ile Leu Gly Gln Thr Val Pro Cys Ser Thr Thr Thr 210 215 220 Ile AsnGly Ser Thr His Val Leu Arg Tyr Thr Val Leu His Arg Gly 225 230 235 240His His Gln Glu Leu Glu Lys Leu Pro Ile His Asn Asp Pro Glu Ser 245 250255 Leu Glu Ser Cys Phe 260 16 445 PRT Rattus norvegicus 16 Met Met AspVal Asn Ser Ser Gly Arg Pro Asp Leu Tyr Gly His Leu 1 5 10 15 Arg SerPhe Leu Leu Pro Glu Val Gly Arg Gly Leu Pro Asp Leu Ser 20 25 30 Pro AspGly Gly Ala Asp Pro Val Ala Gly Ser Trp Ala Pro His Leu 35 40 45 Leu SerGlu Val Thr Ala Ser Pro Ala Pro Thr Trp Asp Ala Pro Pro 50 55 60 Asp AsnAla Ser Gly Cys Gly Glu Gln Ile Asn Tyr Gly Arg Val Glu 65 70 75 80 LysVal Val Ile Gly Ser Ile Leu Thr Leu Ile Thr Leu Leu Thr Ile 85 90 95 AlaGly Asn Cys Leu Val Val Ile Ser Val Cys Phe Val Lys Lys Leu 100 105 110Arg Gln Pro Ser Asn Tyr Leu Ile Val Ser Leu Ala Leu Ala Asp Leu 115 120125 Ser Val Ala Val Ala Val Met Pro Phe Val Ser Val Thr Asp Leu Ile 130135 140 Gly Gly Lys Trp Ile Phe Gly His Phe Phe Cys Asn Val Phe Ile Ala145 150 155 160 Met Asp Val Met Cys Cys Thr Ala Ser Ile Met Thr Leu CysVal Ile 165 170 175 Ser Ile Asp Arg Tyr Leu Gly Ile Thr Arg Pro Leu ThrTyr Pro Val 180 185 190 Arg Gln Asn Gly Lys Cys Met Ala Lys Met Ile LeuSer Val Trp Leu 195 200 205 Leu Ser Ala Ser Ile Thr Leu Pro Pro Leu PheGly Trp Ala Gln Asn 210 215 220 Val Asn Asp Asp Lys Val Cys Leu Ile SerGln Asp Phe Gly Tyr Thr 225 230 235 240 Ile Tyr Ser Thr Ala Val Ala PheTyr Ile Pro Met Ser Val Met Leu 245 250 255 Phe Met Tyr Tyr Gln Ile TyrLys Ala Ala Arg Lys Ser Ala Ala Lys 260 265 270 His Lys Phe Pro Gly PhePro Arg Val Glu Pro Asp Ser Val Ile Ala 275 280 285 Leu Asn Gly Ile ValLys Leu Gln Lys Glu Val Glu Glu Cys Ala Asn 290 295 300 Leu Ser Arg LeuLeu Lys His Glu Arg Lys Asn Ile Ser Ile Phe Lys 305 310 315 320 Arg GluGln Lys Ala Ala Thr Thr Leu Gly Ile Ile Val Gly Ala Phe 325 330 335 ThrVal Cys Trp Leu Pro Phe Phe Leu Leu Ser Thr Ala Arg Pro Phe 340 345 350Ile Cys Gly Thr Ser Cys Ser Cys Ile Pro Leu Trp Val Glu Arg Thr 355 360365 Phe Leu Trp Leu Gly Tyr Ala Asn Ser Leu Ile Asn Pro Phe Ile Tyr 370375 380 Ala Phe Phe Asn Arg Asp Leu Arg Thr Thr Tyr Arg Ser Leu Leu Gln385 390 395 400 Cys Gln Tyr Arg Asn Ile Asn Arg Lys Leu Ser Ala Ala GlyMet His 405 410 415 Glu Ala Leu Lys Leu Ala Glu Arg Pro Glu Arg Pro GluPhe Val Leu 420 425 430 Gln Asn Ala Asp Tyr Cys Arg Lys Lys Gly His AspSer 435 440 445 17 471 PRT Rattus norvegicus 17 Met Glu Ile Leu Cys GluAsp Asn Ile Ser Leu Ser Ser Ile Pro Asn 1 5 10 15 Ser Leu Met Gln LeuGly Asp Gly Pro Arg Leu Tyr His Asn Asp Phe 20 25 30 Asn Ser Arg Asp AlaAsn Thr Ser Glu Ala Ser Asn Trp Thr Ile Asp 35 40 45 Ala Glu Asn Arg ThrAsn Leu Ser Cys Glu Gly Tyr Leu Pro Pro Thr 50 55 60 Cys Leu Ser Ile LeuHis Leu Gln Glu Lys Asn Trp Ser Ala Leu Leu 65 70 75 80 Thr Thr Val ValIle Ile Leu Thr Ile Ala Gly Asn Ile Leu Val Ile 85 90 95 Met Ala Val SerLeu Glu Lys Lys Leu Gln Asn Ala Thr Asn Tyr Phe 100 105 110 Leu Met SerLeu Ala Ile Ala Asp Met Leu Leu Gly Phe Leu Val Met 115 120 125 Pro ValSer Met Leu Thr Ile Leu Tyr Gly Tyr Arg Trp Pro Leu Pro 130 135 140 SerLys Leu Cys Ala Ile Trp Ile Tyr Leu Asp Val Leu Phe Ser Thr 145 150 155160 Ala Ser Ile Met Asn Leu Cys Ala Ile Ser Leu Asp Arg Tyr Val Ala 165170 175 Ile Gln Asn Pro Ile His His Ser Arg Phe Asn Ser Arg Thr Lys Ala180 185 190 Phe Leu Lys Ile Ile Ala Val Trp Thr Ile Ser Val Gly Ile SerMet 195 200 205 Pro Ile Pro Val Phe Gly Leu Gln Asp Asp Ser Lys Val PheLys Glu 210 215 220 Gly Ser Cys Leu Leu Ala Asp Asp Asn Phe Val Leu IleGly Ser Phe 225 230 235 240 Val Ala Phe Phe Ile Pro Leu Thr Ile Met ValIle Thr Tyr Phe Leu 245 250 255 Thr Ile Lys Ser Leu Gln Lys Glu Ala ThrLeu Cys Val Ser Asp Leu 260 265 270 Ser Thr Arg Ala Lys Leu Ala Ser PheSer Phe Leu Pro Gln Ser Ser 275 280 285 Leu Ser Ser Glu Lys Leu Phe GlnArg Ser Ile His Arg Glu Pro Gly 290 295 300 Ser Tyr Ala Gly Arg Arg ThrMet Gln Ser Ile Ser Asn Glu Gln Lys 305 310 315 320 Ala Cys Lys Val LeuGly Ile Val Phe Phe Leu Phe Val Val Met Trp 325 330 335 Cys Pro Phe PheIle Thr Asn Ile Met Ala Val Ile Cys Lys Glu Ser 340 345 350 Cys Asn GluAsn Val Ile Gly Ala Leu Leu Asn Val Phe Val Trp Ile 355 360 365 Gly TyrLeu Ser Ser Ala Val Met Pro Leu Val Tyr Thr Leu Phe Met 370 375 380 LysThr Tyr Arg Ser Ala Phe Ser Arg Tyr Ile Gln Cys Gln Tyr Lys 385 390 395400 Glu Asn Arg Lys Pro Leu Gln Leu Ile Leu Val Asn Thr Ile Pro Ala 405410 415 Leu Ala Tyr Lys Ser Ser Gln Leu Gln Val Gly Gln Lys Lys Asn Ser420 425 430 Gln Glu Asp Ala Glu Gln Thr Val Asp Asp Cys Ser Met Val ThrLeu 435 440 445 Gly Lys Gln Gln Ser Glu Glu Asn Cys Thr Asp Asn Ile GluThr Val 450 455 460 Asn Glu Lys Val Ser Cys Val 465 470 18 460 PRTRattus norvegicus 18 Met Val Asn Leu Gly Asn Ala Val Arg Ser Leu Leu MetHis Leu Ile 1 5 10 15 Gly Leu Leu Val Trp Gln Phe Asp Ile Ser Ile SerPro Val Ala Ala 20 25 30 Ile Val Thr Asp Thr Phe Asn Ser Ser Asp Gly GlyArg Leu Phe Gln 35 40 45 Phe Pro Asp Gly Val Gln Asn Trp Pro Ala Leu SerIle Val Val Ile 50 55 60 Ile Ile Asn Thr Ile Gly Gly Asn Ile Leu Val IleMet Ala Val Ser 65 70 75 80 Met Glu Lys Lys Leu His Asn Ala Thr Asn ThrPhe Leu Met Ser Leu 85 90 95 Ala Ile Ala Asp Met Leu Val Gly Leu Leu ValMet Pro Leu Ser Leu 100 105 110 Leu Ala Ile Leu Tyr Asp Tyr Val Trp ProLeu Pro Arg Tyr Leu Cys 115 120 125 Pro Val Trp Ile Ser Leu Asp Val LeuPhe Ser Thr Ala Ser Ile Met 130 135 140 Asn Leu Cys Ala Ile Ser Leu AspArg Tyr Val Ala Ile Arg Asn Pro 145 150 155 160 Ile Glu His Ser Arg PheAsn Ser Arg Thr Lys Ala Ile Met Lys Ile 165 170 175 Ala Ile Val Trp AlaIle Ser Ile Gly Val Ser Val Pro Ile Pro Val 180 185 190 Ile Gly Leu ArgAsp Glu Ser Lys Val Phe Val Asn Asn Thr Thr Cys 195 200 205 Val Leu AsnAsp Pro Asn Phe Val Leu Ile Gly Ser Phe Val Ala Phe 210 215 220 Phe IlePro Leu Thr Ile Met Val Ile Thr Tyr Phe Leu Thr Ile Tyr 225 230 235 240Val Leu Arg Arg Gln Thr Leu Met Leu Leu Arg Gly His Thr Glu Glu 245 250255 Glu Leu Ala Asn Met Ser Leu Asn Phe Leu Asn Cys Cys Cys Lys Lys 260265 270 Asn Gly Gly Glu Glu Glu Asn Ala Pro Asn Pro Asn Pro Asp Gln Lys275 280 285 Pro Arg Arg Lys Lys Lys Glu Lys Arg Pro Arg Gly Thr Met GlnAla 290 295 300 Ile Asn Asn Glu Lys Lys Ala Ser Lys Val Leu Gly Ile ValPhe Phe 305 310 315 320 Val Phe Leu Ile Met Trp Cys Pro Phe Phe Ile ThrAsn Ile Leu Ser 325 330 335 Val Leu Cys Gly Lys Ala Cys Asn Gln Lys LeuMet Glu Lys Leu Leu 340 345 350 Asn Val Phe Val Trp Ile Gly Tyr Val CysSer Gly Ile Asn Pro Leu 355 360 365 Val Tyr Thr Leu Phe Asn Lys Ile TyrArg Arg Ala Phe Ser Lys Tyr 370 375 380 Leu Arg Cys Asp Tyr Lys Pro AspLys Lys Pro Pro Val Arg Gln Ile 385 390 395 400 Pro Arg Val Ala Ala ThrAla Leu Ser Gly Arg Glu Leu Asn Val Asn 405 410 415 Ile Tyr Arg His GluAsn Glu Arg Val Ala Arg Lys Ala Asn Asp Pro 420 425 430 Glu Pro Gly IleGlu Met Gln Val Glu Asn Leu Glu Leu Pro Val Asn 435 440 445 Pro Ser AsnVal Val Ser Glu Arg Ile Ser Ser Val 450 455 460 19 359 PRT canine 19 MetIle Ser Asn Gly Thr Gly Ser Ser Phe Cys Leu Asp Ser Pro Pro 1 5 10 15Cys Arg Ile Thr Val Ser Val Val Leu Thr Val Leu Ile Leu Ile Thr 20 25 30Ile Ala Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg 35 40 45Leu Arg Ser Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp 50 55 60Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Phe Tyr Gln Leu 65 70 7580 Ser Cys Arg Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser 85 9095 Leu Asp Val Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile 100105 110 Ser Leu Asp Arg Tyr Cys Ala Val Thr Asp Pro Leu Arg Tyr Pro Val115 120 125 Leu Ile Thr Pro Val Arg Val Ala Val Ser Leu Val Leu Ile TrpVal 130 135 140 Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gly TrpAsn Ser 145 150 155 160 Arg Asn Glu Thr Ser Ser Phe Asn His Thr Ile ProLys Cys Lys Val 165 170 175 Gln Val Asn Leu Val Tyr Gly Leu Val Asp GlyLeu Val Thr Phe Tyr 180 185 190 Leu Pro Leu Leu Val Met Cys Ile Thr TyrTyr Arg Ile Phe Lys Ile 195 200 205 Ala Arg Asp Gln Ala Lys Arg Ile HisHis Met Gly Ser Trp Lys Ala 210 215 220 Ala Thr Ile Gly Glu His Lys AlaThr Val Thr Leu Ala Ala Val Met 225 230 235 240 Gly Ala Phe Ile Ile CysTrp Phe Pro Tyr Phe Thr Val Phe Val Tyr 245 250 255 Arg Gly Leu Lys GlyAsp Asp Ala Ile Asn Glu Ala Phe Glu Ala Val 260 265 270 Val Leu Trp LeuGly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr 275 280 285 Ala Thr LeuAsn Arg Asp Phe Arg Thr Ala Tyr Gln Gln Leu Phe Arg 290 295 300 Cys ArgPro Ala Ser His Asn Ala Gln Glu Thr Ser Leu Arg Ser Asn 305 310 315 320Ser Ser Gln Leu Ala Arg Asn Gln Ser Arg Glu Pro Met Arg Gln Glu 325 330335 Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr Ala 340345 350 Pro Arg Gly Ala Thr Asp Arg 355

What is claimed is:
 1. An isolated nucleic acid molecule encoding amammalian 5-HT₄ receptor.
 2. A nucleic acid molecule of claim 1, whereinthe nucleic acid molecule encodes a human 5-HT₄ receptor.
 3. A nucleicacid molecule of claim 1, wherein the nucleic acid molecule is a DNAmolecule.
 4. A DNA molecule of claim 3, wherein the DNA molecule is acDNA molecule.
 5. A DNA molecule of claim 3 wherein the DNA molecule isgenomic DNA.
 6. A nucleic acid molecule of claim 2, wherein the nucleicacid molecule is a DNA molecule.
 7. An isolated DNA molecule encoding amammalian 5-HT₄ receptor having the sequence H₂N—Y—X—COOH wherein Y isthe amino acid sequence beginning at amino acid 1 and ending at aminoacid 359 of FIG. 1 (SEQ ID No. 2) and wherein X is an amino acidsequence encoding the carboxy terminal region of the receptor.
 8. Anisolated nucleic acid molecule of claim 7, wherein X is the amino acidsequence beginning at amino acid 360 and ending at amino acid 387 ofFIG. 1 (SEQ ID NO. 2).
 9. An isolated nucleic acid molecule of claim 7,wherein X is the amino acid sequence beginning at amino acid 360 andending at amino acid 406 of FIG. 2 (SEQ ID NO. 4).
 10. An isolatednucleic acid molecule of claim 7, wherein Y is encoded by the nucleotidesequence from nucleotide 101 to nucleotide 1177 of FIG. 1 (SEQ ID NO.1).
 11. An isolated nucleic acid molecule of claim 8, wherein X isencoded by the nucleotide sequence from nucleotide 1178 to nucleotide to1261 of FIG. 1 (SEQ ID NO. 1).
 12. An isolated nucleic acid molecule ofclaim 9, wherein X is encoded by the nucleotide sequence from nucleotide1127 to nucleotide 1267 of FIG. 2 (SEQ ID NO. 3).
 13. A vectorcomprising a cDNA molecule of claim
 4. 14. A plasmid vector of claim 13.15. A vector of claim 13 adapted for expression in a bacterial cellwhich comprises regulatory elements necessary for expression of the cDNAencoding a 5-HT₄ receptor in the bacterial cell operatively linked tothe cDNA encoding the 5-HT₄ receptor as to permit expression thereof.16. A vector of claim 13 adapted for expression in a yeast cell whichcomprises the regulatory elements necessary for the expression of thecDNA encoding a 5-HT₄ receptor in the yeast cell operatively linked tothe cDNA encoding the 5-HT₄ receptor as to permit expression thereof.17. A vector of claim 13 adapted for expression in an insect cell whichcomprises the regulatory elements necessary for expression of the cDNAencoding a 5-HT₄ receptor in the insect cell operatively linked to thecDNA encoding the 5-HT₄ receptor as to permit expression thereof.
 18. Avector of claim 13 adapted for expression in a mammalian cell whichcomprises the regulatory elements necessary for expression of the cDNAencoding a 5-HT₄ receptor in the mammalian cell operatively linked tothe cDNA encoding the 5-HT₄ receptor as to permit expression thereof.19. A plasmid of claim 14 adapted for expression in a mammalian cellwhich comprises the regulatory elements necessary for expression of theDNA in the mammalian cell operatively linked to the DNA encoding a 5-HT₄receptor as to permit expression thereof.
 20. A plasmid of claim 19designated pcEXV-S10-87 (ATCC Accession No. 75390).
 21. A plasmid ofclaim 19 designated pcEXV-S10-95 (ATCC Accession No. 75391).
 22. Aplasmid of claim 19 designated pBluescript-hS10 (ATCC Accession No.75392).
 23. A mammalian cell comprising the plasmid of claim
 14. 24. Amammalian cell of claim 23, wherein the mammalian cell is an LM (tk−)cell.
 25. A nucleic acid probe comprising a nucleic acid molecule of atleast 15 nucleotides capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a mammalian 5-HT₄ receptor.
 26. A nucleic acid probe comprisinga nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a unique sequence included within thesequence of a nucleic acid molecule encoding a human 5-HT₄ receptor. 27.The nucleic acid probe of claim 25, wherein the nucleic acid is DNA. 28.The nucleic acid probe of claim 26, wherein the nucleic acid is DNA. 29.A mixture of nucleic acid probes in accordance with claim 25, suchprobes having sequences which differ from one another at predefinedpositions.
 30. An antisense oligonucleotide having a sequence capable ofspecifically binding to a mRNA molecule encoding a mammalian 5-HT₄receptor so as to prevent translation of the mRNA molecule.
 31. Anantisense oligonucleotide capable of specifically binding to a mRNAmolecule encoding a human 5-HT₄ receptor so as to prevent translation ofthe mRNA molecule.
 32. An antisense oligonucleotide of claim 30comprising chemical analogs of nucleotides.
 33. A mixture of antisenseoligonucleotides according to claim 30, such oligonucleotides havingsequences which differ from one another at predefined positions.
 34. Amethod for detecting expression of a mammalian 5-HT₄ receptor, whichcomprises obtaining RNA from cells or tissue, contacting the RNA soobtained with a nucleic acid probe of claim 25 under hybridizingconditions, detecting the presence of any mRNA hybridized to the probe,the presence of mRNA hybridized to the probe indicating expression ofthe mammalian 5-HT₄ receptor, and thereby detecting the expression ofthe mammalian 5-HT₄ receptor.
 35. A method for detecting expression of ahuman 5-HT₄ receptor, which comprises obtaining RNA from cells ortissue, contacting the RNA so obtained with a nucleic acid probe ofclaim 26 under hybridizing conditions, detecting the presence of anymRNA hybridized to the probe, the presence of mRNA hybridized to theprobe indicating expression of the human 5-HT₄ receptor, and therebydetecting the expression of the human 5-HT₄ receptor.
 36. A method ofdetecting expression of a mammalian 5-HT₄ receptor in a cell or tissueby in situ hybridization, which comprises contacting the cell or tissuewith a nucleic acid probe of claim 25 under hybridizing conditions,detecting the presence of any mRNA hybridized to the probe, the presenceof mRNA hybridized to the probe indicating expression of a mammalian5-HT₄ receptor, and thereby detecting the expression of a mammalian5-HT₄ receptor.
 37. A method of detecting expression of a human 5-HT₄receptor in a cell or tissue by in situ hybridization, which comprisescontacting the cell or tissue with a nucleic acid probe of claim 26under hybridizing conditions, detecting the presence of any mRNAhybridized to the probe, the presence of mRNA hybridized to the probeindicating expression of a human 5-HT₄ receptor, and thereby detectingthe expression of the human 5-HT₄ receptor.
 38. A method of isolatingfrom a gene library a gene encoding a receptor other than the 5-HT₄receptor which comprises contacting the library under hybridizingconditions with a probe of claim 27 and isolating any gene to which theprobe hybridizes.
 39. A method of claim 38, which additionally comprisessimultaneously contacting the DNA comprising the library underhybridizing conditions with a second nucleic acid probe comprising asequence capable of hybridizing to a DNA sequence of the complementarystrand of the DNA of the gene to which the first probe hybridizes,treating any gene sequence to which both probes hybridized so as toproduce multiple copies of the gene sequence, isolating the amplifiedgene sequence and using the isolated gene sequence as a probe to isolatefrom a gene library the gene to which the amplified DNA sequencehybridizes.
 40. The gene isolated by the method of claim 38 or
 39. 41. Asynthetic gene which comprises the isolated nucleic acid molecule ofclaim 1 and at least one regulatory element attached thereto so as toincrease the number of RNA molecules transcribed from the syntheticgene.
 42. A synthetic gene which comprises the isolated nucleic acidmolecule of claim 1 and at least one regulatory element attached theretoso as to decrease the number of RNA molecules transcribed from thesynthetic gene.
 43. An isolated mammalian 5-HT₄ receptor protein. 44.The receptor protein of claim 43, wherein the mammalian 5-HT₄ receptorprotein is a human 5-HT₄ receptor.
 45. A method of preparing a mammalian5-HT₄ receptor of claim 43, which comprises inducing cells to expressthe mammalian 5-HT₄ receptor and recovering the mammalian 5-HT₄ receptorfrom the resulting cells.
 46. A method of preparing a mammalian 5-HT₄receptor protein of claim 43, which comprises inserting a nucleic acidmolecule encoding the mammalian 5-HT₄ receptor in a suitable vector,inserting the resulting vector in suitable host cell and recovering themammalian 5-HT₄ receptor produced by the resulting cell.
 47. A method ofpreparing a human 5-HT₄ receptor of claim 44, which comprises inducingcells to express the human 5-HT₄ receptor and recovering the human 5-HT₄receptor from the resulting cells.
 48. A method of preparing a human5-HT₄ receptor protein of claim 44, which comprises inserting a nucleicacid molecule encoding the human 5-HT₄ receptor in a suitable vector,inserting the resulting vector in suitable host cell and recovering thehuman 5-HT₄ receptor produced by the resulting cell.
 49. An antibodydirected to a mammalian 5-HT₄ receptor or to a protein fragment of themammalian 5-HT₄ receptor.
 50. An antibody directed to a human 5-HT₄receptor or to a protein fragment of the human 5-HT₄ receptor.
 51. Anantibody of claim 49, wherein the antibody is a monoclonal antibody. 52.An antibody of claim 50, wherein the antibody is a monoclonal antibody.53. A monoclonal antibody of claim 51, wherein the antibody is directedto an epitope of a mammalian cell-surface 5-HT₄ receptor and having anamino acid sequence substantially the same as the amino acid sequence ofa cell-surface epitope of the mammalian 5-HT₄ receptor.
 54. A monoclonalantibody of claim 52, wherein the antibody is directed to an epitope ofa human cell-surface 5-HT₄ receptor and having an amino acid sequencesubstantially the same as the amino acid sequence for a cell-surfaceepitope of the human 5-HT₄ receptor.
 55. A pharmaceutical compositioncomprising an amount of a substance effective to alleviate theabnormalities resulting from overexpression of a human 5-HT₄ receptorand a pharmaceutically acceptable carrier.
 56. A pharmaceuticalcomposition comprising an amount of a substance effective to alleviateabnormalities resulting from underexpression of a human 5-HT₄ receptorand a pharmaceutically acceptable carrier.
 57. A pharmaceuticalcomposition comprising an effective amount of an oligonucleotide ofclaim 31 effective to reduce expression of a human 5-HT₄ receptor bypassing through a cell membrane and specifically binding with mRNAencoding a human 5-HT₄ receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable hydrophobic carriercapable of passing through a cell membrane.
 58. A pharmaceuticalcomposition claim 57, wherein the nucleotide is coupled to a substancewhich inactivates mRNA.
 59. A pharmaceutical composition of claim 58,wherein the substance which inactivates the mRNA is a ribozyme.
 60. Apharmaceutical composition of claim 58, wherein the pharmaceuticallyacceptable hydrophobic carrier capable of passing through a cellmembrane comprises a structure which binds to a transporter specific fora selected cell type and is thereby taken up by the cells of theselected cell type.
 61. A pharmaceutical composition which comprises anamount of the antibody of claim 50 effective to block binding ofnaturally occurring substrates to a human 5-HT₄ receptor and apharmaceutically acceptable carrier.
 62. A transgenic nonhuman mammalwhich comprises a nucleic acid molecule of claim
 1. 63. A transgenicnonhuman mammal whose genome comprises a nucleic acid molecule of claim1 so placed as to be transcribed into antisense mRNA complementary tomRNA encoding a human 5-HT₄ receptor and which hybridizes to mRNAencoding a human 5-HT₄ receptor thereby reducing its translation. 64.The transgenic nonhuman mammal of claim 62, wherein the nucleic acidmolecule further comprises an inducible promoter.
 65. The transgenicnonhuman mammal of claim 62 or 64 wherein the nucleic moleculeadditionally comprises tissue specific regulatory elements.
 66. Thetransgenic non-human mammal of 62 wherein the transgenic non-humanmammal is a mouse.
 67. A method for determining the physiologicaleffects of varying the levels of expression of a human 5-HT₄ receptorwhich comprises producing a transgenic non-human mammal whose levels ofexpression of a human 5-HT₄ receptor can be varied by use of aninducible promoter.
 68. A method for determining the physiologicaleffects of expressing varying levels of a human 5-HT₄ receptor whichcomprises producing a panel of transgenic non-human mammals eachexpressing a different amount of a human 5-HT₄ receptor.
 69. A methodfor determining whether a compound not known to be capable ofspecifically binding to a human 5-HT₄ receptor can specifically bind tothe human 5-HT₄ receptor, which comprises contacting a mammalian cellcomprising a plasmid adapted for expression in a mammalian cell whichplasmid further comprises DNA which expresses a human 5-HT₄ receptor onthe cell's surface with the compound under conditions permitting bindingof ligands known to bind to a human 5-HT₄ receptor, detecting thepresence of any compound bound to the human 5-HT₄ receptor, the presenceof bound compound indicating that the compound is capable ofspecifically binding to the human 5-HT₄ receptor.
 70. The method ofclaim 70, wherein the mammalian cell is a non-neuronal cell.
 71. Amethod of screening compounds to identify drugs which interact with, andspecifically bind to, a human 5-HT₄ receptor on the surface of a cell,which comprises contacting a mammalian cell which comprises a plasmidadapted for expression in a mammalian cell which plasmid furthercomprises DNA which expresses a human 5-HT₄ receptor on the cell'ssurface with a plurality of compounds, determining those compounds whichbind to the human 5-HT₄ receptor expressed on the cell surface of themammalian cell, and thereby identifying compounds which interact with,and specifically bind to, the human 5-HT₄ receptor.
 72. The method ofclaim 71, wherein the mammalian cell is a non-neuronal cell.
 73. Amethod for determining whether a compound not known to be capable ofspecifically binding to a human 5-HT₄ receptor can specifically bind toa human 5-HT₄ receptor, which comprises preparing a cell extract frommammalian cells, which comprise a plasmid adapted for expression in amammal, which plasmid further comprises DNA which expresses a human5-HT₄ receptor on the cell's surface, isolating a membrane fraction fromthe cell extract, incubating the compound with the membrane fractionunder conditions permitting binding of ligands known to bind to thehuman 5-HT₄ receptor, detecting the presence of any bound compound, andthereby determining whether the compound is capable of specificallybinding to the human 5-HT₄ receptor.
 74. The method of claim 73, whereinthe mammalian cell is a non-neuronal cell.
 75. A method for screeningcompounds to identify drugs that interact with, and specifically bindto, a human 5-HT₄ receptor, which comprises preparing a cell extractfrom mammalian cells, which comprise a plasmid adapted for expression ina mammalian cell which plasmid further comprises DNA which expresses ahuman 5-HT₄ receptor on the cell's surface, isolating a membranefraction from the cell extract, incubating the membrane fraction with aplurality of compounds, determining those compounds which interact withand bind to the human 5-HT₄ receptor, and thereby identifying compoundswhich interact with, and specifically bind to, the human 5-HT₄ receptor.76. The method of claim 75, wherein the mammalian cell is a non-neuronalcell.
 77. A method for identifying a compound which is not known to becapable of binding to the human 5-HT₄ receptor activates the human 5-HT₄receptor on the surface of a mammalian cell or prevents a ligand whichdoes so, which comprises contacting the mammalian cell which cellcomprises a plasmid adapted for expression in the mammalian cell suchplasmid further comprising DNA which expresses the human 5-HT₄ receptoron the cell surface of the mammalian cell with the compound underconditions permitting activation of blockade of a functional response,determining whether the compound activates the human 5-HT₄ receptor orprevents a ligand which does so, and thereby identifying the compound asa compound which interacts with, and activates the human 5-HT₄ receptoror prevents the activation of the human 5-HT₄ receptor by a ligand whichdoes so.
 78. The method of claim 77, wherein the mammalian cell is anon-neuronal cell comprising the cellular components necessary toproduce a second messenger and wherein the determination of whether thecompound activates of blocks the activation of the human 5-HT₄ comprisesdetecting the change in the concentration of the second messenger. 79.The method of claim 78, wherein the second messenger is cyclic AMP(cAMP).
 80. The method of claim 78, wherein the non-neuronal cell is aCOS-7 cell.
 81. A method of claim 78, wherein the second messenger is aninositol phosphate metabolite.
 82. The method of claim 78, wherein thesecond messenger is intracellular calcium.
 83. A compound identified bythe method of claim 69, 73 or
 77. 84. A pharmaceutical composition of adrug identified by the method of claim 71 or
 75. 85. A method fordetecting the presence of a human 5-HT₄ receptor on the surface of acell, which comprises contacting the cell with an antibody of claim 50,under conditions that permit binding of the antibody to the receptor,detecting the presence of any of the antibody bound to the cell, andthereby the presence of a human 5-HT₄ receptor on the surface of thecell.
 86. A method for treating an abnormal condition related to anexcess of activity of a human 5-HT₄ receptor, which comprisesadministering a patient an amount of a pharmaceutical composition ofclaim 84, effective to reduce 5-HT₄ activity as a result of naturallyoccurring substrate binding to and activating the 5-HT₄ receptor. 87.The method of treating abnormalities which are alleviated by an increasein the activity of a 5-HT₄ receptor, which comprises administering apatient an amount of a pharmaceutical composition of claim 84, effectiveto increase the activity of the 5-HT₄ receptor thereby alleviatingabnormalities resulting from abnormally low receptor activity.
 88. Amethod for diagnosing a predisposition to a disorder associated with theexpression of a human 5-HT₄ receptor allele which comprises: a.obtaining DNA from subjects suffering from a disorder; b. performing arestriction digest of the DNA with a panel of restriction enzymes; c.electrophoretically separating the resulting DNA fragments on a sizinggel; d. contacting the gel with a nucleic acid probe capable ofspecifically hybridizing to DNA encoding a human 5-HT₄ receptor andlabelled with a detectable marker; e. detecting the labelled bands whichhave hybridized to the DNA encoding 5-HT₄ receptor, labelled with thedetectable marker to create a unique band pattern specific to the DNA ofsubjects suffering with the disorder; f. preparing DNA for diagnosis bysteps a-e; g. comparing the unique band pattern specific to the DNA ofpatients suffering from the disorder from step e and DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.
 89. The method of claim 88, wherein a disorder isassociated with the expression of a specific human 5-HT₄ receptor alleleis diagnosed.
 90. A method of identifying a substance capable ofalleviating the abnormalities resulting from overexpression of a human5-HT₄ receptor which comprises administering a substance to thetransgenic non-human mammal of claim 67 or 68, and determining whetherthe substance alleviates the physical and behavioral abnormalitiesdisplayed by the transgenic nonhuman mammal as a result ofoverexpression of the human 5-HT₄ receptor.
 91. A method of identifyinga substance capable of alleviating the abnormalities resulting fromunderexpression of a human 5-HT₄ receptor, which comprises administeringa substance to the transgenic mammal of claim 62, and determiningwhether the substance alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofunderexpression of the human 5-HT₄ receptor.
 92. A method of treatingabnormalities in a subject, wherein the abnormality is alleviated by thereduced expression of a human 5-HT₄ receptor which comprisesadministering to a subject an effective amount of the pharmaceuticalcomposition of claim 55, 57, 83 or 84 effective to reduce expression ofthe human 5-HT₄ receptor.
 93. A method of treating abnormalitiesresulting from underexpression of a human 5-HT₄ receptor which comprisesadministering to a subject an amount of a pharmaceutical composition ofclaim 56, 83 or 84 effective to alleviate abnormalities resulting fromunderexpression of the human 5-HT₄ receptor.